10,574 research outputs found
Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology
No full textImmunosuppression is required for BK viremia and polyomavirus BK-associated nephropathy (PVAN) in kidney transplants (KTs), but the role of viral determinants is unclear. We examined BKV noncoding control regions (NCCR), which coordinate viral gene expression and replication. In 286 day-matched plasma and urine samples from 129 KT patients with BKV viremia, including 70 with PVAN, the majority of viruses contained archetypal (ww-) NCCRs. However, rearranged (rr-) NCCRs were more frequent in plasma than in urine samples (22 vs. 4%; P > 0.001), and were associated with 20-fold higher plasma BKV loads (2.0 x 10(4)/ml vs. 4.4 x 10(5)/ml; P > 0.001). Emergence of rr-NCCR in plasma correlated with duration and peak BKV load (R(2) = 0.64; P > 0.001). This was confirmed in a prospective cohort of 733 plasma samples from 227 patients. For 39 PVAN patients with available biopsies, rr-NCCRs were associated with more extensive viral replication and inflammation. Cloning of 10 rr-NCCRs revealed diverse duplications or deletions in different NCCR subregions, but all were sufficient to increase early gene expression, replication capacity, and cytopathology of recombinant BKV in vitro. Thus, rr-NCCR BKV emergence in plasma is linked to increased replication capacity and disease in KTs
In vitro and in vivo characterization of the cytomegalovirus and polyomavirus BK specific immune response
No full textDuring my PhD thesis several aspects of the interaction of Cytomegalovirus
and Polyomavirus BK with the host’s immune system were examined (see list
of publications). The overall aim was to compare immune response in healthy
individuals and kidney transplant recipients with or without viral replication.
In healthy individuals, Polyomaviruses BK and JC infect 80% and 60%,
respectively. For CMV seroprevalences may reach up to 80%. Intermittent
virus shedding in urine is observed for BKV in 7%, JCV in 19% and CMV in
0%. However, no virus replication in plasma was detected. Posttransplant,
mainly due to prolonged immune suppression the amount and function of
CMV- and BKV-specific T-cells is impaired. Calcineurin inhibitors lead to a
direct reduction of INFγ production of virus-specific T-cells, whereas antiproliferative
immunosuppressives reduce the expansion capacity in a dosedependent
manner. This may be a major reason for uncontrolled virus
replication.
The humoral response reflects the amount of recent antigen exposure and
does not directly indicate protection from virus replication. Virus-specific
cellular immune responses would probably allow to assessing the risk of
future replication.
Overall the importance of CMV and BKV specific T-cells posttransplant in
controlling virus replication was examined. For both viruses we could
calculate a protective threshold of virus-specific T-cells. CMV-pp65 specific
CD4 T-cells above 0.03% were significantly associated with no CMV
replication during the next eight weeks. Additionally, below this cut-off, CMV
seropositive recipients developed more often GCV-resistant CMV replication.
During BKV replication, patients with more than 69 BKV-LT specific T-cells
per 1 Mio PBMCs were significantly more often showing decreasing BKV
loads in plasma. As virus-specific T-cells seem to be crutial in reducing virus replication, and
reduction of immune suppression harbours the risk of acute rejection, a
booster vaccine could be a new therapeutic option. A booster vaccine could
probably elevate the amount of virus-specific T-cells above a critical threshold
of protection from disease, despite effects of immune suppression.
We tried to identify immunodominant regions with the CMV pp65 and BKV LT
proteins. We used a combined approach of computer prediction algorithms
and experimental verification. Epitope mapping of BKV LT with computer
prediction revealed several clusters, which could be immunodominant and
also potentially be processed and recognized in various HLA backgrounds.
The identified cluster regions were synthesised as 15 and 25mers. Expansion
and re-stimulation with predicted epitopes could so far confirm the HLA A and
B-specific prediction of single 9mers covered by the larger 15 and 25mer
sequences. However, other HLA types need to be tested for direct stimulation
and expansion potential of the predicted epitopes. Additionally, tetramer
staining should be performed for verification.
Based on this research, we will be able to improve current immune monitoring
and probably a high-specific peptide-based vaccine against BKV LT could be
developed and be used to increase the amount of BKV-LT specific T-cells.
Another potentially therapeutic agent could be the blockade of PD1 ligand.
PD1 expression in chronic virus infection lead to impaired CD8+ T-cell
function. CMV-specific CD4 T-cells treated with an inhibitory antibody against
PD1 ligand, and thereby activating CD4+ T-cells, lead to a increase of the
expansion capacity. We have shown, that the anti-PD1 ligand antibody
increases various cytokines. This could be also tested for BK virus.
Measurement of virus-specific T-cells may replace serological assays in the
future, due to a better correlation to effective antiviral control, which can be
used as monitoring tool during infection and post-vaccination
Characterizing determinants of BK Polyomavirus-specific immune response
No full textBK polyomavirus (BKPyV) is one of now 13 human polyomavirus (HPyV) species detected in humans. BKPyV is only known to infect humans and seroprevalence rates of more than 90% have been reported in adult populations around the world. Following primary infection, BKPyV persists in the renourinary tract without causing any disease as evidenced by urinary shedding in 5% - 10% of healthy immunocompetent blood donors.
In immunocompromised persons, however, BKPyV can cause significant diseases whereby uncontrolled high-level replication may lead to organ invasive pathologies in kidneys, bladder, lungs, vasculature, and the central nervous system. The most consistently found diseases are BKPyV-associated hemorrhagic cystitis (BKPyVHC) in 5%-20% allogeneic hematopoietic stem cells transplant patients, and BKPyV-associated nephropathy (BKPyVAN) in 1%-15% of kidney transplant patients. BKPyVHC is highly symptomatic with pain, anemic bleeding, and increased mortality. BKPyVAN is asymptomatic except for progressive renal failure and premature return to dialysis. Both entities are characterized by high-level viral replication i.e. with urine BKPyV loads of 8-10 log10 Geq/mL, plasma BKPyV loads often above 4 log10 Geq/mL, and an allogeneic constellation between the virus-infected host cell and the available T-cell effectors. Despite these similarities, the clinical manifestations are strikingly different suggesting relevant, but experimentally undefined differences in pathogenesis. Thus, BKPyVHC typically occurs within 4 weeks after allogeneic HSCT and is confined to the bladder, and typically without kidney involvement. By contrast, BKPyVAN is diagnosed around 3-6 months after kidney transplantation and confined to the kidney allograft without causing cystitis. Although high-level BKPyV replication should be formally amenable to antiviral drug treatment, no effective and BKPyV-specific antiviral therapy is currently available. Therefore, a better understanding of the immune alteration in both diseases has been deemed essential to identify patients at risk and to develop prophylactic, preemptive and therapeutic strategies.
The currently recommended strategy for BKPyVAN is to screen kidney transplant patients for BKPyV replication and to promptly reduce immunosuppressive therapy in those with significant replication to facilitate mounting of BKPyV-specific T cell responses and thereby preventing progression to disease. This manoeuver has been linked to expanding BKPyV-specific T cell responses in the peripheral blood of kidney transplant patients. However, this approach may place patients at risk for acute rejection episodes that predispose equally well to premature kidney transplant failure. Although the clinical feasibility of reducing immunosuppression and curtailing BKPyV replication has been shown to be effective in prospective cohort studies for many, but not all of kidney transplant patients, this approach has not been possible in allogeneic HSCT patients because of concurrent or imminent graft-versus host disease. Thus, there are significant gaps in the current understanding of the BKPyV– host interaction in the normal host and in the allogeneic setting, which need to be investigated for a more effective and safer management of these significant viral complications.
In this thesis, the interaction of BKPyV and the immune response has been approached from two different angles. In the first project, potential mechanisms of BKPyV immune evasion were studied. Here, we focused on a small accessory protein called agnoprotein encoded as a leader protein in the late viral early region (LVGR). Although HPyV genomes overall show a very similar genome organization, agnoproteins are only found in the genomes of BKPyV and JCPyV that have a kidney tropisms, but not in any of the other 11 presumably non-renotropic HPyVs. We hypothesized that agnoprotein could play a role in immune evasion by downregulating HLA expression. The effects of agnoprotein were studied on HLA class I and II expression in vitro by flow cytometry following transfection of primary human renal tubular epithelial cells, which are the viral target of BKPyV-associated nephropathy. In addition, transfected human UTA-6 cells were studied as well as UTA-6 cells bearing a tetracycline-regulated agnoprotein. As control, the effects were compared with the ICP47 protein of Herpes simplex virus-1, which has been previously reported to effectively down-regulate HLA class I. Although both viral proteins share some similarities at the protein level, our results showed that BKPyV agnoprotein did not down-regulate HLA class I or class II molecules. Also, there was not inhibitory effect on the increase of HLA-class I or class-II surface expression following exposure to interferon-. By contrast, ICP47 reduced HLA class I surface expression, but not class II. We also evaluated effects of agnoprotein on virus epitope-specific T-cell killing by 51Chromium release assay, however no interference could be observed. We concluded that agnoprotein did not contribute to these types of HLA-dependent immune evasion processes. However, further investigations are needed to understand if agnoprotein could contribute to viral immune escape by other mechanisms.
In the second project, we aimed at better characterizing BKPyV-specific CD8 T cell immunity targeting epitopes encoded in the early viral gene region (EVGR). Selected coding sequences of the BKPyV EVGR were submitted to two web-based computer algorithms (SYFPEITHI, IEDB) in order to predict immunodominant 9mer epitopes presented by 14 frequent HLA-class I molecules. For an experimental confirmation, 97 different 9mer epitopes were chemically synthesized and tested in 42 healthy individuals. A total of 39 epitopes could be confirmed by interferon- ELISpot assay in at least 30% of healthy individuals. Interestingly, most of the 9mer epitopes appeared to cluster in short amino acid stretches, and some 9mer could be presented by more than one HLA class I allele as expected for immunodominant domains. HLA-specific presentation was demonstrated by 9mer- MHC-I streptamers for 21/39 (54%) epitopes. The 9mer dependent T-cell killing by 51Chromium release assay and the CD107a surface detection indicated that the 9mer epitopes could be recognized by cytotoxic T-cells. Moving to a clinically relevant situation, 13 9mer epitopes could be validated in 19 kidney transplant patients protected from, or recovering from, BKPyV viremia. The results suggest that, pending further corroboration in larger patient populations, novel 9mer epitopes can be identified, which are associated with CD8 T cell control of BKPyV replication. Thus the identified immunodominant 9mer T-cell epitopes could be further developed for clinical assays to better predict the risk and the recovery of BKPyV diseases, help guiding immunosuppression reduction, and to develop specific adoptive T-cell therapy or vaccine responses to prevent or treat BKPyV-associated disease
Desenvolvimento e aplicação de um sistema celular repórter para herpes simplex virus e padronização de uma PCR quantitativa para poliomavírus BK
No full textDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2014.Pacientes imunodeprimidos podem apresentar infecções virais com evolução rápida, sintomatologias atípicas graves e muitas vezes fatais, sendo fundamental um diagnóstico precoce para estabelecimento do tratamento efetivo, redução da toxicidade e da resistência aos antivirais. HSV-1, HSV-2 e poliomavírus BK são vírus de importância clínica para imunodeprimidos e podem levar a rejeição de órgãos em transplantados. Assim, o objetivo deste trabalho foi desenvolver um sistema celular repórter, utilizando a proteína fluorescente GFP, para HSV-1 e 2 e implantar uma qPCR utilizando amostras clínicas de pacientes transplantados renais para detecção de poliomavírus BK. O sistema celular repórter foi construído através da transfecção de células Vero com o vetor pZsGreen1-1 ligado ao promotor ICP10 (F3R3 e F4R3) da RR1 do HSV-2. A regulação da expressão da GFP via ICP10 é dependente da infecção viral e acontece por meio da proteína viral transativadora VP16 e de fatores celulares Oct-1 e HCF-1. A efetividade do sistema foi avaliada por infecção viral e pela aplicação de antivirais (Aciclovir, ácido gálico, convalotoxina e extrato de Uncaria sp.) e candidatos antivirais inativos (Extrato de Passiflora edulis e derivados de cardenolídeos). O sistema repórter F4R3 ZsGreen1-1 expressou GFP em função da infecção por HSV-1 e 2, a qual foi detectada por microscopia de fluorescência e/ou citometria de fluxo. Em análise por citometria de fluxo, a fluorescência do sistema repórter correlacionou-se diretamente com os títulos virais (MOI de 4,0 x10-3 a 3,3 x10-4, ou seja, 1 partícula viral a cada 250 a 3000 células), o sistema manteve a capacidade de expressão da GFP na presença de agentes sem propriedade antiviral e não expressou fluorescência quando tratado com antivirais. O sistema F4R3 ZsGreen1-1 mostrou-se um sistema funcional com possíveis aplicações para diagnóstico clínico, para elaboração de testes de resistência aos antivirais e para a pesquisa de novos medicamentos. A qPCR para poliomavírus BK foi implantada utilizando amostras de DNA cedidas pelo HEMOSC com iniciadores dirigidos para o antígeno T viral. O limite de detecção foi de 18 cópias genômicas/ reação com quantificações variando entre 9,8 x 105 a 6,7 x 107 cópias genômicas/ mL. A qPCR foi efetiva para análises de amostras clínicas e apresentou limite de detecção suficiente para avaliação de risco de nefropatia em transplantados renais.Abstract : Immunosuppressed patients can present viral infections with fast evolution, severe atypical symptomatologies and often fatal, being essential the early diagnosis for the establishment of effective treatments, reduction of toxicity and development of resistance to antiviral. HSV-1, HSV-2 and polyomavirus BK are virus of clinical importance for immunosuppresed and can lead to the rejection of transplanted organs. Therefore, the aim of this work was to develop a reporter cellular system, using the fluorescent protein GFP, for HSV-1 and 2, and deploy a qPCR using clinical samples from patients submitted to renal transplant, for the detection of polyomavirus BK. The reporter cellular system was constructed through the transfection of Vero cells with the vector pZsGreen1-1 connected to the promoter ICP10 (F3R3 and F4R3) of the RR1 of the HSV-2. The regulation of the expression of GFP via ICP10 is dependent of the viral infection and happens through the viral transactivating protein VP16, and the cellular factors Oct-1 and HCF-1. The effectivity of the system was evaluated by viral infection and through the application of antiviral (Acyclovir, gallic acid, convalotoxina and extract of Uncaria sp.) and inactive antiviral candidate (Extract of Passiflora edulis and derivatives cardenolide). The reporter system F4R3 ZsGreen1-1 expressed GFP as a function of the infection for HSV-1 and 2, which was detected by fluorescence microscopy and/or flow cytometry. In flow cytometry, the fluorescence of the reporter system was directly correlated with virus titers (MOI 4,0 x10-3 to 3,3 x10-4, that is, 1 viral particle to each 250 to 3000 cells), the system maintained the ability to GFP expression in the presence of agents without antiviral property and no expressed fluorescence when treated with antivirals. The system F4R3 ZsGreen1-1 revealed a functional system with possible applications for clinical diagnosis, elaboration of tests of resistance to the antiviral and for new drugs research. The qPCR to polyomavirus BK was deployed using DNA samples provided by HEMOSC with primers directed to the viral antigen T. The limit of detection was 18 genome copies /reaction with quantification ranging between 9.8 x 105 to 6.7 x 107 genome copies /mL. The qPCR was effective for the analyses of clinical samples and presented enough sensitivity for risk evaluation of nephropathy in renal transplant
Nothing Gained by Overcrowding: Sir Raymond Unwin
No full textIn his 1912 pamphlet for the Garden Cities and Town Planning Association Nothing Gained by Overcrowding, Raymond Unwin set out in detail the lessons learnt from his formidable practical experience in the design and layout o f housing: at New Earswick from 1902, Letchworth gard en c ity from 1905, and most significantly at Hampstead garden Suburb, where the ‘artisans’ quarter’ 1907–9 was probably his masterwork o f spatial design. His interest in minimising the length o f paved road to number o f houses served, and ‘greening’ the ubiquitous mechanistic bye-law suburb o f the late 19th century provided motivation for defining a general theory o f design, which underpinned Garden City principles. Nothing Gained by Overcrowding emerged as a principle which was to have a revolutionary impact on housing and urban form over the next 50 years.
Unwin’s theory had developed with his work, but the origins can be found in two earlier and less well known publications. ‘On the building o f houses in the Garden City’ was written for the first international conference o f the Garden City Association, held in September 1901. The following year he published the Fabian Society Tract Cottage Plans and Common Sense, in which he took first principles, ‘shelter, comfort, privacy’, and drew out general criteria and specific standards. Housing had to be freed from the bye-law straitjacket. This would sweep away ‘back yards, back alleys and abominations ... too long screened by that wretched prefix back’.
Republished here for the first time together, with an introductory essay by Dr Mervyn Miller, these three papers make clear the development o f Raymond Unwin’s theories o f planning and housing, theories which were among the most influential o f the 20th Century
Paper in Architecture: Research by design, engineering and prototyping
No full textPaper is a fascinating material that we encounter every day in different variants: tissues, paper towels, packaging material, wall paper or even fillers of doors. Despite radical changes in production technology, the material, which has been known to mankind for almost two thousand years, still has a natural composition, being made up of fibres of plant origin (particularly wood fibres). Thanks to its unique properties, relatively high compression strength and bending stiffness, low production costs and ease of recycling, paper is becoming more and more popular in many types of industry.
Mass-produced paper products such as special paper, paperboard, corrugated cardboard, honeycomb panels, tubes and L- and U-shapes are suitable for use as a building material in the broad sense of these words — i.e., in design and architecture. Objects for everyday use, furniture, interior design elements and partitions are just a few examples of things in which paper can be employed. Temporary events such as festivals, exhibitions or sporting events like the Olympics require structures that only need to last for a limited period of time. When they are demolished after a few days or months, their leftovers can have a significant impact on the local environment.
In the context of growing awareness of environmental threats and the efforts undertaken by local and international organisations and governments to counter these threats, the use of natural materials that can be recycled after their lifespan is becoming increasingly widespread.
Paper and its derivatives fascinate designers and architects, who are always looking for new challenges and trying to meet the market’s demands for innovative and proecological solutions. Being a low-cost and readily available material, paper is suited to the production of emergency shelters for victims of natural and man-made disasters, as well as homeless persons.
In order to gain a better understanding of paper’s potential in terms of architecture, its material properties were researched on a micro, meso and macro level. This research of the possible applications of paper in architecture was informed by two main research questions:
What is paper and to what extent can it be used in architecture?
What is the most suitable way to use paper in emergency architecture?
To answer the first research question, fundamental and material research on paper and paper products had to be conducted. The composition of the material, production methods and properties of paper were researched. Then paper products with the potential to be used in architecture were examined. The history of the development of paper and its influence on civilisation helped the author gain a better understanding of the nature of this material, which we encounter in our lives every day. Research on objects for everyday use, furniture, pavilions and architecture realised in the last 150 years allowed the author to distinguish various types of paper design and paper architecture. Analysis of realised buildings in which paper products were used as structural elements and parts of the building envelope resulted in a wide array of possible solutions. Structural systems, types of connections between the various elements, impregnation methods and the functionalities and lifespan of different types of buildings were systematised. The knowledge thus collected allowed the author to conduct a further exploration of paper architecture in the form of designs and prototypes.
To answer the second research question, the analysed case studies were translated into designs and prototypes of emergency shelters.
During the research-by-design, engineering and prototyping phases, more than a dozen prototypes were built. The prototypes differed in terms of structural systems, used materials, connections between structural elements, impregnation methods, functionality and types of building. The three versions of the Transportable Emergency Cardboard House project presented in the final chapter form the author’s final answer to the second research question.
Paper will never replace traditional building materials such as timber, concrete, steel, glass or plastic. It can, however, complement them to a significant degree
Effect of protein leaking BK-F PMMA-based hemodialysis on plasma pentosidine levels.
No full textBACKGROUND: Advanced glycation end-products (AGEs) are now considered to contribute to the middle molecule toxicity of uremia and, because they are not cleared by conventional low-flux hemodialysis, alternative strategies are needed to improve their removal. METHODS: In a prospective cross-over trial involving 18 adult chronic hemodialysis subjects, we evaluated the intradialytic removal and the long-term effect on predialysis levels of Protein-bound (PBPe) and Free (FPe) pentosidine by high-pore, protein-leaking BK-F Polymethylmethacrylate-based hemodialysis (BK-F-HD), by comparing it to hemodialysis using low-flux dialyzers (LF-HD). RESULTS: A single BK-F-HD session removed more PBPe, but not FPe, than LF-HD. Long-term BK-F-HD was associated with a significant decrease in pre-dialysis PBPe, FPe, and albumin (17.7 +/- 20.8, 25.3 +/- 17.3 and 8.0 +/- 3.3%, p<0.01) and no change in body mass index and protein catabolic rate, compared to LF-HD. Multiple stepwise regression analysis identified C-reactive Protein (CRP) (standardized beta coefficient=-0.629), pre-dialysis levels in LF-HD (beta=0.452) and dialysis vintage (beta=0.428) as significant determinants of BK-F-induced changes in predialysis PBPe, and predialysis FPe and PBPe levels in LF-HD as significant determinants of BK-F-induced changes in predialysis FPe (beta=0.720 and 0.286, respectively). CONCLUSIONS: Our study shows that long-term standard diffusive hemodialysis with BK-F membrane reduces predialysis PBPe and FPe levels by comparison with LF-HD, largely due to a greater intradialytic clearance of PBPe. Serum albumin is also reduced without any associated changes in nutritional status markers. The study also suggests that the effect of BK-F-HD in lowering PBPe levels is modulated by the body burden of pentosidine and is blunted or even lost in the presence of elevated CRP levels
JC and BK polyomavirus-like particles as targets of innate and adaptive humoral immunity
No full textJC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) were identified as the first of now more than 12 human polyomaviruses (HPyVs). The average JCPyV and BKPyV seroprevalence rates in adults are 70% and 90%, respectively. After asymptomatic infection both viruses persist in the renourinary tract. In fact, asymptomatic viruria is detectable in one-third of general population. However, in immunocompromised patients, JCPyV and BKPyV replication may progress to significant diseases. Hence, JCPyV can cause progressive multifocal leukoencephalopathy (PML) in patients with HIV-AIDS, malignancies or autoimmune diseases under immunosuppressive treatment. BKPyV can be a cause of polyomavirus-associated nephropathy (PyVAN) in kidney transplant recipients or hemorrhagic cystitis (PyVHC) after allogeneic hematopoietic stem cell transplantation. Due to more frequent application of immunosuppression, the risk of developing these diseases has increased in the last few decades. The risk of PML development is estimated to be 100-fold higher for JCPyV-seropositive patients in comparison to JCPyV-seronegatives. Most cases of PyVAN and PyVHC have been tested positive for BKPyV at the moment of disease diagnosis. Unfortunately, there is no specific antiviral therapy against any of these HPyV diseases. Thus, current strategies to avert PyVAN or PyVHC aim at identifying patients with BKPyV viremia and reducing immunosuppression. Similar strategies for PML have not been effective, since JCPyV viremia is usually not detected prior to or at the diagnosis of disease. The fate of BKPyV and JCPyV virus-like particles (VLPs) was examined in an animal model corresponding to primary viremia in non-immune host. Radioactively labeled VLPs were used to assess blood decay, organ, and hepatocellular distribution of ligand, and non-labeled VLPs to examine cellular uptake by immunohisto- and cytochemistry. Rapid distribution of both BKPyV and JCPyV VLPs to the liver was observed, with lesser uptake in kidney and spleen. Liver uptake was predominantly observed in LSECs. Blood half-life and tissue distribution of both wild-type JCPyV VLPs and two mutant JCPyV VLPs (L55F and S269F), lacking sialic acid binding affinity, were similar, indicating involvement of non-sialic acid receptors in cellular uptake. We concluded that LSECs very effectively cleared a large fraction of blood-borne BKPyV and JCPyV VLPs, indicating a central role of these cells in early removal of polyomavirus from the circulation. Moreover, we observed that a subpopulation of endothelial cells in kidney, the main organ of polyomavirus persistence, showed selective and rapid uptake of VLPs, suggesting a role in viremic organ tropism (Simon-Santamaria et al., p. 54). Giving the increasing clinical need to reliably determine JCPyV and BKPyV IgG levels in patients at risk, we first reviewed and optimized serological tools for JCPyV and BKPyV IgG detection including virus-like particle (VLP)-based ELISA. We demonstrated that although no statistically significant differences in intraassay and interassay variability were revealed for JCPyV serology of 400-fold diluted sera from healthy donors, qualitative differences were seen in the identification of the individual JCPyV serostatus. The cause of discordance for approximately 10% of sera resulted from a low IgG activity close to the cutoff of the assay. Therefore we standardized the ELISA using reference serum for normalization. Moreover, we developed a preadsorption assay with cutoff of 35% reduction of the JCPyV IgG activity after preincubation with JCPyV VLPs. Importantly, we excluded BKPyV antibody cross-reactivity by testing JCPyV IgG positive sera in preadsorption assay using BKPyV VLPs. In conclusion, we showed that VLP-based ELISA with normalization can serve as a reliable tool for JCPyV IgG serology. Additionally, the preadsorption assay can help with unequivocal determination of JCPyV serostatus for samples with low IgG levels. (Kardas et al., p. 72). We also normalized this VLP-based ELISA for BKPyV IgG detection and showed that for seroepidemiology studies, normalized JCPyV and BKPyV IgG ELISA at 1:200 serum dilution provides optimal sensitivity and specificity with the lowest false-positive and false-negative rate. However, for individual risk assessment, 100-, 200-, and 400-fold dilutions combined with preadsorption for low-reactive sera might be the most appropriate (Kardas et al., p. 82). This improved ELISA was used to investigate JCPyV and BKPyV specific antibody levels in several clinical studies: (1) one case of PML patient where positive JCPyV IgG status was compatible with other PML-indicating symptoms (Kurmann et al., p. 90); (2) one case of PyVAN caused by JCPyV rather than BKPyV, as confirmed by JCPyV IgG/IgM positive and BKPyV IgG/IgM negative results (Lautenschlager et al., 99); (3) one case of PyVHC patient after allogeneic hematopoietic stem cell transplantation where increasing BKPyV IgG activities were in line with progression of BKPyV viremia (Koskenvuo et al., p. 106). Further, by serological testing of 122 immunocompetent and 63 immunocompromised patients we demonstrated that the BKPyV IgG level is age-dependent, with the highest values between 20 and 30 years (Schmidt et al., p. 119). In another study we compared serological outcomes of ELISA utilizing two different antigens in terms of prognostic value in prostate cancer development. To accomplish this we utilized improved ELISA for BKPyV IgG activity to both BKPyV VLPs and BKPyV LTag. Testing of 226 patients undergoing radical prostatectomy for primary prostate cancer revealed that BKPyV VP1 serostatus, in contrast to BKPyV LTag, has no prognostic value in prostate cancer progression (Keller et al., p. 125). In conclusion, we provided a new input into knowledge about tropism and clearance of polyomaviruses from blood. Moreover, we established a reliable and sensitive VLP-based assay for specific detection of JCPyV and BKPyV IgG and IgM. Serostatus based on ELISA results was compatible with other symptoms of BKPyV- and JCPyV-related diseases
Polyomavirus BK-specific cellular immune response in Kidney transplant recipients
No full textPolyomavirus BK is an emerging pathogen in KT recipients. New potent
immunosuppressive drugs promote reactivation and replication of BKV and progression
towards PVAN. PVAN occurs in up to 10% of the KT recipients with a graft loss in up to
80% of the cases. New potent immunosuppressive drugs, as MMF) and FK506 are risk factors
for developing PVAN. As no proven antiviral drugs are available, the only therapy of choice
is the reduction of immunosuppressiva in order to regain BKV-replication control (H. H.
Hirsch, M. Dickenmann, S. Binggeli, J. Steiger, Schweiz Med Forum 2004; 4:538–541).
BKV-specific cellular and humoral immune response is not well characterized. Recent
findings have shown that BKV-seropositive patients prior to transplantation are not protected
from BKV-replication. In contrast, BKV-specific cellular immune response correlates with
the diagnosis of PVAN (P. Comoli, S. Binggeli, F. Ginevri, H. H. Hirsch, Transplant
Infectious Disease Jun 2006; 8(2):86-94, Review).
The aim of this study was to investigate the interplay of BKV-specific immune response
and BKV-replication in blood samples of KT recipients. We examined the BKV-specific
immune response by ELISpot assay in KT. PBMC of KT recipients were stimulated with
BKV LT-antigen and BKV-VP1 peptide libraries. The BKV-specific immune response was
measured by the detection of IFN-γ by ELISpot assay. From the results of a pilot study with
eight patients we were able to deduce that the dynamics of viral-replication rather than the
viral load correlates with a protective immune response (S. Binggeli, A. Egli, M.
Dickenmann, I. Binet, J. Steiger, H. H. Hirsch, American Journal of Transplantation, Sep
2006; 6(9):2218-9).
To corroborate this previous observation the BKV-specific cellular immunity in 42 KT
recipients and 10 HB were tested. The KT patients were divided into two groups: patient
group 1 with an increasing or stable viral load (inc/hi)1 and patient group 2 with a decreasing
viral load or after resolved PVAN (dec)2. Indeed patients in group 2 showed a significantly
higher immune response upon stimulation with BKV-LT and BKV-VP1 than patients in
group 1 (P=0.003, P=0.001, respectively, Wilcoxon, two-sided). Detailed analysis revealed a
cut-off of >69 SFU/Mio PBMC for BKV LT-antigen, but not for BKV VP1, with
significantly more KT patients from group 2 (dec) than from group 1 (inc/hi). This cut-off has
to be validated in a prospective study and also analyzed whether such a cut-off can be used for
immunosuppressive reduction guidance.
BKV-specific cell expansion was tested in a short-term culture in the presence of either
BKV-LT or -VP1. After 9-day culture, PBMC were restimulated with BKV-LT or -VP1 and
the responses were then compared with responses to direct stimulation (without prior
cultivation). BKV-LT and -VP1 specific cellular immune responses were significantly higher
after 9-day cultivation than after direct stimulation (P=0.002, P=0.003, respectively,
Wilcoxon, two sided).
Due to high sequence homology between JCV and BKV, JCV-LT and -VP1 overlapping
peptide pools were used to test PBMC-cross recognition. JCV-LT and -VP1 responses were
significantly lower than BKV-mediated response (P=0.008, P<0.001, respectively, Wilcoxon,
two-sided). Comparison of JCV- and BKV-specific responses after 9-day culture revealed that
the BKV-VP1 response was significantly higher than the JCV-VP1 (P=0.016, Wilcoxon, two
sided), but no significant difference was observed for LT-antigen (S. Binggeli, A. Egli, S.
Schaub, I. Binet, M. Mayr, J. Steiger, H. H. Hirsch, American Journal of Transplantation, Mar
2007; 7:1-9).
Agnoprotein, a late viral protein, is highly expressed upon infection. We investigated
whether agnoprotein is able to induce a BKV-specific immune response and whether it may
serve as a diagnostic marker. Immunostaining revealed that agnoprotein was highly expressed
in the cytoplasm of infected cells and was only seen in combination with BKV-LT which is
located in the nucleus. Interestingly, BKV-agnoprotein specific cellular and humoral immune
responses were scarcely detected in HB or KT recipients. There are only few published
studies concerning BKV-agnoprotein, and further investigations are necessary to fully
understand the function of agnoprotein during infection. (D. Leuenberger, P. A. Andresen, R.
Gosert, S. Binggeli, E. H. Ström, S. Bodaghi, C Hanssen Rinaldo, H. H. Hirsch, Clinical and
Vaccine Immunology, Aug 2007; 14(8): 959-968).
As no antiviral treatment is available for BKV, the only therapy is the reduction of
immunosuppressive drugs in order to regain immunological control over BKV-replication and
PVAN. However reduction of immunosuppressants upon PVAN diagnosis bears the risk of
rejection or inflammatory response to BKV. It is difficult to distinguish between these two
outcomes because specific markers are yet lacking. Therefore, it is pivotal to record the
clinico-pathological course of the KT patient in order to correctly diagnose the problem as the
therapies are completely different. Measuring the BKV-specific cellular immune response
may support and complement other markers, such as PCR analysis and biopsies, to better
distinguish between rejection and BKV-specific immune response. (S. Schaub, M. Mayr, A.
Egli, S. Binggeli, B. Descoeudres, J. Steiger, M. J. Mihatsch, H. H. Hirsch, Nephrology
Dialysis Transplantation, Aug 2007; 22(8): 2386-90).
Finding the optimal immunosuppressive drug level is crucial for preventing rejection
(under-immunosuppressed) and viral replication (over-immunosuppressed). Our current study
showed a cut-off level of 6.65 ng/ml FK506 drug level in blood, dividing those KT patients
with and without BKV-replication control (ROC-curve: AUC=0.897, sensitivity=78%,
specificity=86%). If this cut-off is validated by a well designed prospective study, it may
serve as a guideline to administrate the optimal drug level. (S. Binggeli, 2007, current results).
BKV-specific epitopes have received considerable attention in the last five years. We
started with the epitope mapping in a kidney patient with the most common HLA-type: HLAA*
01, HLA-B*08. First screening of BKV-LT revealed ten 15aa long peptides with
immunogenic potential. Three of these ten peptides were further investigated for crossrecognition
with the homologous JCV-peptides. Even though response to the three JCVpeptides
was lower, cellular immune response could be clearly detected. It needs further
investigation to find more BKV-specific epitopes and also to test the ability of CD8+ T-cells
to kill BKV-antigen presenting cells. (S. Binggeli, 2007, current results)
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