Le polyomavirus BK est un virus très prévalent qui demeure normalement en phase de latence dans l’uroépithélium sans entrainer de complications. Chez les greffés rénaux, il peut cependant se réactiver et mener à une néphropathie pouvant nuire à la survie du greffon. L’immunité du receveur est la pierre angulaire de la prévention et du traitement de cette néphropathie, puisque le seul traitement démontré efficace est une diminution de l’immunosuppression. Cependant, une augmentation non spécifique de l’immunité augmente également le risque de rejet. Notre objectif était donc d’adapter et de valider un protocole transférable en clinique d’immunothérapie adoptive antivirale nous permettant de produire des lignées de lymphocytes T BKvirus spécifiques à partir du sang de patients greffés virémiques, afin de prévenir et traiter ces néphropathies. Nous avons tout d’abord comparé les lignées cellulaires produites à partir de donneurs sains à celles de patients immunosupprimés soumis à une immunosuppression chronique. Par la suite, nous avons adapté le protocole en ajoutant une stimulation à l’aide de cellules dendritiques afin de maximiser l’expansion cellulaire, le statut de différentiation et la spécificité. Bien que les lignées étaient polyclonales, elles n’ont pas démontré de potentiel alloréactif in vivo et in vitro, et ce, malgré une persistance et une prolifération in vivo. Nous avons donc élaboré un protocole qui est prêt à être transféré en étude clinique de phase I/II et qui pourrait nous permettre de prévenir et traiter la néphropathie associée au polyomavirus BK, sans augmenter le risque de rejet. / More than 75% of the population has been exposed to BK polyomavirus and carries latent virus in the uroepithelium without any complications. However, it can reactivates in kidney transplant recipients (KTR) and lead to a nephropathy affecting graft survival. Recipient anti-viral immunity is the cornerstone of BK-virus associated nephropathy prevention and treatment and thus, reduction of immunosuppression is the only well-accepted treatment. Adoptive immunotherapy is a promising solution to this problem, allowing a specific T cell mediated response against this virus without the alloreactive risk. It was demonstrated efficacious for other viral infections in immunocompromised hosts but it has not been used in this specific context. Our objective was to adapt and validate a clinical-compliant protocol to obtain BK-specific T cell lines from viremic KTR and to compare their expansion, differentiation and specificity to ones obtained from healthy donors. Although comparable specificity and differentiation status, cell expansions form KTR were not systematically sufficient for a therapeutic dose. The addition of a stimulation with dendritic cells improved cell expansion in addition to favors a central memory phenotype and refined BKspecificity. Despite polyclonality, T cell lines didn’t demonstrated alloreactivity in a chromium release assay and in vivo. Furthermore, T cell lines could persist and proliferates in vivo. This protocol is ready for a phase I/II clinical trial. This opens the possibility to solve the current conundrum and treat PVAN without increasing rejection risk.

INTRODUÇÃO: O BKPyV está associado à inflamação e perda da função do enxerto em pacientes transplantados renais. Nos pacientes transplantados renais, aproximadamente 40% dos receptores desenvolvem viruria pelo BKPyV em até 3 meses e 20% desenvolvem viremia em até 1 ano pós-transplante. Os pacientes que desenvolvem viremia têm o risco de evolução para nefropatia associada ao BKPyV (NABKPyV), com prevalência em torno de 1 a 10%, e evolução para perda do enxerto renal bastante variável, de 0 a 100%, dependendo dos estudos e das intervenções realizadas. Embora a vigilância de replicação do BKPyV seja recomendada, existem diferenças de metodologia e periodicidade entre as recomendações publicadas. Adicionalmente, tem sido discutida a importância do cut-off de viremia para o manejo clínico desses pacientes na prevenção de evolução para nefropatia. Os objetivos primários deste estudo foram determinar a prevalência de decoy cell na urina, viremia e viremia sustentada pelo BKPyV e NABKPyV, nos receptores de transplante renal do Serviço de Transplante Renal do HCFMUSP, e os possíveis fatores de risco associados à presença a viremia sustentada pelo BKPyV e NABKPyV. MÉTODOS: Trata-se de um estudo de coorte prospectivo no qual foram incluídos todos os receptores e os doadores de transplante renal intervivos submetidos a transplante de agosto de 2010 a dezembro de 2011. Todos os participantes foram avaliados no momento imediato pré-transplante e os receptores foram monitorados para detecção de viremia de BKPyV e desenvolvimento de NABKPyV durante o período de até 2 anos pós-transplante. Os receptores colheram amostras de urina mensalmente, durante o primeiro ano, e a cada 3 meses durante o segundo ano pós-transplante para a pesquisa de viruria (realizada por decoy cell e/ou q-PCR). A detecção de viruria indicava o início de monitorização mensal de viremia por q-PCR, viremia era mantida até obtenção de três amostras de viremia negativas consecutivas. A detecção da primeira viremia positiva deveria ser confirmada por uma segunda amostra colhida após intervalo de duas semanas; se o exame repetido confirmasse a viremia positiva, os pacientes eram submetidos à biópsia renal percutânea para investigação de NABKPyV. RESULTADOS: No período do estudo foram realizados 326 transplantes e foram incluídos 246 pacientes. A prevalência de viruria foi de 36,9%, a de viremia 22,3% e a de nefropatia 3,2%. O tempo médio entre o transplante e a viruria positiva pela decoy cell foi de 7,2 meses, entre o transplante e a viremia positiva de 7,6 meses, e entre o transplante e o diagnóstico de NABKPyV de 8,5 meses. O único fator de risco encontrado para viremia sustentada e para nefropatia foi gênero masculino. O valor de cut-off de viremia que melhor discrimina a evolução para NABKPyV foi 44.955 cópias/mL. CONCLUSÕES: As prevalências de viruria, viremia e nefropatia foram semelhantes às reportadas na literatura. O gênero masculino foi o único fato de risco encontrado para viremia sustentada e nefropatia. O valor de cut-off de viremia que melhor discrimina o risco de evolução para nefropatia foi maior que o valor usualmente recomendado pela literatura, que é de 10.000 cópias/mL / INTRODUCTION: BKPyV is associated with inflammation and loss of graft function in kidney transplant patients. In kidney transplantation, approximately 35-47% of recipients develop viruria by BKPyV within 3 months post-transplantation, and 20% develop viremia within one year post-transplantation. Patients who develop viremia are at risk of progression to BKPyV-associated nephropathy (BKPyVAN), with prevalence around 1 to 10%, and a quite variable prevalence of progression to kidney graft loss, ranging from 0 to 100%, depending on the studies and the interventions. Although BKPyV surveillance is recommended, there are differences in methodology and frequency between published recommendations. In addition, the importance of viremia cutoff for clinical management of these patients in the prevention of progression for nephropathy has been discussed. The objectives of this study were to determine the prevalence of decoy cell in urine, BKPyV viremia, BKPyV sustained viremia, and BKPyVAN, in kidney transplant recipients of HCFMUSP Kidney Transplant Service. Additionally, the aim was to determine the possible risk factors associated with the presence of BKPyV sustained viremia and BKPyVAN. METHODS: This is a prospective cohort study. From August 2010 to December 2011, all recipients and donors of kidney transplant who underwent transplantation were enrolled. All participants were evaluated at immediate pre-transplant and recipients were monitored for detection of BKPyV viremia and development of BKPyVAN for up to two years post-transplantation. All recipients collected urine samples monthly during the first year and every three months during the second year post-transplant for viruria screening (performed by decoy cell and/or q-PCR). Viruria detection indicated the initiation of monthly viremia monitoring by q-PCR. Viremia was maintained until three consecutive negative viremia samples were obtained. The detection of the first positive viremia should be confirmed by a second sample collected after a two-week interval. If repeated examination confirmed positive viremia, the patients underwent percutaneous kidney biopsy to investigate BKPyVAN. RESULTS: During the study period, 326 transplants were performed and 246 patients were included. The prevalence of viruria, viremia, and nephropathy was, respectively, 36.9%, 22.3%, and 3.2%. The mean time between transplantation and positive viruria by decoy cell was 7.2 months, between transplantation and positive viremia was 7.6 months, and between transplantation and diagnosis of BKPyVAN was 8.5 months. The only risk factor for sustained viremia and nephropathy was male. Viremia cutoff value that best discriminates the progression to BKPyVAN was 44,955 copies/mL. CONCLUSIONS: The prevalence of viruria, viremia, and nephropathy were similar to those reported in the literature. Male was the only risk factor found for sustained viremia and nephropathy. Viremia cutoff value that best discriminates the risk of progression to nephropathy was greater than the value usually recommended in the literature, which is 10,000 copies/mL

INTRODUÇÃO: O BKPyV está associado à inflamação e perda da função do enxerto em pacientes transplantados renais. Nos pacientes transplantados renais, aproximadamente 40% dos receptores desenvolvem viruria pelo BKPyV em até 3 meses e 20% desenvolvem viremia em até 1 ano pós-transplante. Os pacientes que desenvolvem viremia têm o risco de evolução para nefropatia associada ao BKPyV (NABKPyV), com prevalência em torno de 1 a 10%, e evolução para perda do enxerto renal bastante variável, de 0 a 100%, dependendo dos estudos e das intervenções realizadas. Embora a vigilância de replicação do BKPyV seja recomendada, existem diferenças de metodologia e periodicidade entre as recomendações publicadas. Adicionalmente, tem sido discutida a importância do cut-off de viremia para o manejo clínico desses pacientes na prevenção de evolução para nefropatia. Os objetivos primários deste estudo foram determinar a prevalência de decoy cell na urina, viremia e viremia sustentada pelo BKPyV e NABKPyV, nos receptores de transplante renal do Serviço de Transplante Renal do HCFMUSP, e os possíveis fatores de risco associados à presença a viremia sustentada pelo BKPyV e NABKPyV. MÉTODOS: Trata-se de um estudo de coorte prospectivo no qual foram incluídos todos os receptores e os doadores de transplante renal intervivos submetidos a transplante de agosto de 2010 a dezembro de 2011. Todos os participantes foram avaliados no momento imediato pré-transplante e os receptores foram monitorados para detecção de viremia de BKPyV e desenvolvimento de NABKPyV durante o período de até 2 anos pós-transplante. Os receptores colheram amostras de urina mensalmente, durante o primeiro ano, e a cada 3 meses durante o segundo ano pós-transplante para a pesquisa de viruria (realizada por decoy cell e/ou q-PCR). A detecção de viruria indicava o início de monitorização mensal de viremia por q-PCR, viremia era mantida até obtenção de três amostras de viremia negativas consecutivas. A detecção da primeira viremia positiva deveria ser confirmada por uma segunda amostra colhida após intervalo de duas semanas; se o exame repetido confirmasse a viremia positiva, os pacientes eram submetidos à biópsia renal percutânea para investigação de NABKPyV. RESULTADOS: No período do estudo foram realizados 326 transplantes e foram incluídos 246 pacientes. A prevalência de viruria foi de 36,9%, a de viremia 22,3% e a de nefropatia 3,2%. O tempo médio entre o transplante e a viruria positiva pela decoy cell foi de 7,2 meses, entre o transplante e a viremia positiva de 7,6 meses, e entre o transplante e o diagnóstico de NABKPyV de 8,5 meses. O único fator de risco encontrado para viremia sustentada e para nefropatia foi gênero masculino. O valor de cut-off de viremia que melhor discrimina a evolução para NABKPyV foi 44.955 cópias/mL. CONCLUSÕES: As prevalências de viruria, viremia e nefropatia foram semelhantes às reportadas na literatura. O gênero masculino foi o único fato de risco encontrado para viremia sustentada e nefropatia. O valor de cut-off de viremia que melhor discrimina o risco de evolução para nefropatia foi maior que o valor usualmente recomendado pela literatura, que é de 10.000 cópias/mL / INTRODUCTION: BKPyV is associated with inflammation and loss of graft function in kidney transplant patients. In kidney transplantation, approximately 35-47% of recipients develop viruria by BKPyV within 3 months post-transplantation, and 20% develop viremia within one year post-transplantation. Patients who develop viremia are at risk of progression to BKPyV-associated nephropathy (BKPyVAN), with prevalence around 1 to 10%, and a quite variable prevalence of progression to kidney graft loss, ranging from 0 to 100%, depending on the studies and the interventions. Although BKPyV surveillance is recommended, there are differences in methodology and frequency between published recommendations. In addition, the importance of viremia cutoff for clinical management of these patients in the prevention of progression for nephropathy has been discussed. The objectives of this study were to determine the prevalence of decoy cell in urine, BKPyV viremia, BKPyV sustained viremia, and BKPyVAN, in kidney transplant recipients of HCFMUSP Kidney Transplant Service. Additionally, the aim was to determine the possible risk factors associated with the presence of BKPyV sustained viremia and BKPyVAN. METHODS: This is a prospective cohort study. From August 2010 to December 2011, all recipients and donors of kidney transplant who underwent transplantation were enrolled. All participants were evaluated at immediate pre-transplant and recipients were monitored for detection of BKPyV viremia and development of BKPyVAN for up to two years post-transplantation. All recipients collected urine samples monthly during the first year and every three months during the second year post-transplant for viruria screening (performed by decoy cell and/or q-PCR). Viruria detection indicated the initiation of monthly viremia monitoring by q-PCR. Viremia was maintained until three consecutive negative viremia samples were obtained. The detection of the first positive viremia should be confirmed by a second sample collected after a two-week interval. If repeated examination confirmed positive viremia, the patients underwent percutaneous kidney biopsy to investigate BKPyVAN. RESULTS: During the study period, 326 transplants were performed and 246 patients were included. The prevalence of viruria, viremia, and nephropathy was, respectively, 36.9%, 22.3%, and 3.2%. The mean time between transplantation and positive viruria by decoy cell was 7.2 months, between transplantation and positive viremia was 7.6 months, and between transplantation and diagnosis of BKPyVAN was 8.5 months. The only risk factor for sustained viremia and nephropathy was male. Viremia cutoff value that best discriminates the progression to BKPyVAN was 44,955 copies/mL. CONCLUSIONS: The prevalence of viruria, viremia, and nephropathy were similar to those reported in the literature. Male was the only risk factor found for sustained viremia and nephropathy. Viremia cutoff value that best discriminates the risk of progression to nephropathy was greater than the value usually recommended in the literature, which is 10,000 copies/mL

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