The Study Of Epidemiological Surveillance And Specific Cellular Immune Response Of BK Virus Infection In Renal-transplant Recipients

Objection:BK virus (BKV) belongs to the family Polyomaviridae, is infects up to90%of thegeneral population. However, significant clinical manifestations are rare and limited toindividuals with impaired immune functions. Nephropathy associated with the BKV(BKVN) has emerged as an important cause of allograft failure in renal-transplantrecipients. BKV causes BKVN in1-10%of renal transplant recipients, with graft loss inover50%of cases. This is likely due to the development of better immunosuppressivedrugs in the last two decades, which has lead to a decreased rate of acute rejection in renaltransplant patients but an increase in the incidence of BKVN.BK virus infection can be diagnosed by PCR amplification which dectecting BKVDNA in urine or plasma (viruria or viremia), and real-time PCR techniques can monitorthe load viral load more precisely to divide the progress of viral infection and predicte theBKVN. The gold standard diagnosis of BKVN from a specificity perspective is still a renalbiopsy, with confirmation of BK virus presence in renal allograft tissue.Currently, although the risk factors of BKV infection is still not clear, but BKVN andstrong immunosuppression almost occurred at the same time. This phenomenon impiledthat the cellular immunity play an important role. Therefore, most scholars believe thatimmunesuppression is the most important factor of BKV infection recurrence. However,the research of immune state monitoring of BKV infection in renal transplant recipients is rare up to now. Therefore, this subject based on previous research about epidemiology ofBKV infection in Organ Transplantation Institution of PLA309Hospital. We carry out aprospective randomized study to reveal the immune mechanism of BKV infection whichthrough monitoring virus specific cellular immune in renal transplantation recipients. Inorder to provide immunology theoretical basis that can guide monitor and therapy BKVinfection and BKVN after renal transplantation.Method：1. Inclusion criteria of subjects: Allograft renal transplant recipients in our center wererandomly enrolled in this study according inclusion criteria.2. Collection period and detection methods of specimens: Urine specimens andperipheral blood (PB) specimens were obtained before transplantation and every threemonths after surgery. For persistent unknown elevation of Serum creatinine, if existent atany period of time after transplantation, biopsy （under the guidance of B-US) andpathological diagnosis of the renal graft (general staining&immunohistochemistry) shouldbe performed.3. Grouping: Subjects were categorized into three groups: viruria positive, viremiapositive, histologically confirmed BKVN.4. BKV load test: DNA was extracted from EDTA-anticoagulated plasma, serum, orurine by digestion with proteinase K and purification on silica columns before elution withdouble-distilled water, according to the protocol of the manufacturer of the kit. BKV DNAload of urine and blood samples was detected using real-time PCR assay. The primer pairwas5’-AGAACTGCTCCTCAATGGATG-3’ and5’-AGCTGCCCCTGGACACTCT-3’.For BKV specific detection, we used the Taqman fluorescent probesTGCTCTTGAAGCATATGAAGATGGCCCCA to obtain BK virus–specific fragment.Viruria and viremia was urine and blood BKV DNA load>103copies/ml, histologicaldiagnosis was performed by IHC using SV40BKV T-antigen.5. BKV Specific Cellular Immune Response test: Peripheral mononuclear blood cells (PBMC) were isolated from peripheral venous blood. Lyophilized overlapping peptidepools representing BKV proteins VP1, VP2, VP3, and st and LT antigens were createdaccording to primary aminoacid sequences. By using10-color flow cytometry and newlycreated overlapping peptide pools of five BKV antigens (VP1,VP2, VP3, large T antigen,and small t antigen).6. Other data collection: A breakdown of gender, age, type of kidney donor,post-transplant diabetes mellitus (PTDM), acute rejection, pneumonia in the early periodfollowing surgery, immunoinduction therapy, immunosuppression regimen (single orcombination) was offered for analysis.7. Statistics: Data input and data collection were performed using Excel2010,statistical analysis by SPSS16.0, and graphic plotting by GraphPad Prism5. Normaldistributed measurement data were expressed as Mean±SD (x±s), whereas Non-normaldistributed data presented as medium. Statistical methods: f-test and nonparametric testwere mainly performed for measurement data; and rank-sum test, chi-square test andLogistic regression and correlation analysis for count data. P<0.05was considered asstatistical significance.Result：1. The median BKV DNA load of BKVN VS. non-BKVN recipients in both urine andplasma had significance(P＜0.05). The viruria sensitivity is100%, sepectifictiy is57.7%(P=0.03), and the viremia sensitivity is100%, sepectifictiy is82.9%(P=0.0002). Weregraded as viral load≧105copies/ml in plasma or≥107copies/ml in urine as the bestdiscriminant cut-off value to predict the disease and to identify patients at risk ofdeveloping BKVAN. The positive cut-off value of urine’s positive predictive value (PPV+)is26.3%, negative predictive vaule (PPV-) is95.7%, and The positive cut-off value ofplasma’s positive predictive value (PPV+) is83.3%, negative predictive vaule (PPV-) is98.8%.2. Clincal observation for210recipients underwent an allograft transplantation, urine BKV DNA positive (viruria)92cases,43.8%, with a median urine BKV DNA load of4.32×105copies/ml [5.73×103～9.44×109copies/ml]; blood BKV DNA positive (viremia)46cases,21.9%, with a median blood BKV DNA load of5.55×103copies/ml[3.28×103~11.38×106copies/ml]; histologically confirmed BKVN7cases,3.3%.Incidence of viruria and viremia peaked at the6~9th month after transplantation.3. According to analysis of effect of immunosuppression regimen on BKV infection,FK506was found a risk factor of viremia after transplantation (r=0.232, P=0.012), whereasCsA was demonstrated to have an inverse relationship with viremia (r=-0.232, P=0.012),both with statistical significance. No association was found between variousimmunoinduction regimen (ATG/IL-2R/non-induction) and post-surgery BKV infection(P>0.05). BKV infection after transplantation can be considered a risk factor ofpost-operation infection and progression of BKV. While recipient sex, donor type,complication of DM, acute rejection, pneumonia in the early period following surgery werenot statistically relevant to BKV infection (P>0.05).4. The lowest serum creatinine after transplantation within3moths in BK viremiarecipients was108.6±31.5μmol/L, while blood BKV DNA was positive, their serumcreatinine was187.5±85.6μmol/L (P<0.05). But not found statistically relevant in BKviruria recipients.5. T-cell responses to BKV antigens (VP1、VP2、VP3、LT and st) were CD4dominated with a low incidence of patients with detectable BKV-specific CD8T cells(>90%). Frequency of IL-2/IFN-γ+/TNF-α+CD4+T cell respond was declinedsignificantly with stimulating VP1, V2and VP3in virtro when Viremia and BKVN(P<0.05). LT and st were not found statistically relevant in the same situation (P>0.05).6. Structural proteins VP1, V2and VP3elicited significantly higher frequencies ofIL-2/IFN-γ+/TNF-α+CD4+T cell respond stimulating in virtro when Viremia andBKVN campared with regulatory proteins st and LT antigens. And VP1and VP3significantly higher than VP2(P<0.05). 7. Sustain viremia recipientss progressed for BKVN easily, and Frequency ofIL-2/IFN-γ+/TNF-α+CD4+T cell respond was declined significantly with stimulating allfive BKV specifictiy proteins in virtro. Sustain viruria recipientss were not found thisphenomenon.Conclusion1. The kit for testing the BK virus, which could be used to predict BKVAN aftertransplantation when the serum BKV load exceeds105copies/ml or the urine BKV loadexceeds107copies/ml. So the two indexes can be as a diagnostic BKVN progressthreshold value or positive index.2. The peak times of BKV infection was apparently three to nine months aftertransplantation, suggesting the importance of monitoring urine or blood BKV DNA loadsin post-transplantation patients closely during this period in order to reduce BKVAN aftertransplantation, especially for sustain viruria or viremia recipients．3. The recipients who received FK506would be independent risk factor of evolvingBK viremia and BKVN after transplantation. Ruducing or changing this durg for high riskrecipients is a effictive measure for BKVN progress after transplantation. The recipientswho are BK viremia before transplantation would be general clinical independent riskfactor, monitoring and screening for those recipients and donors in preoperative andpostoperative is one of the effective measures to prevent and reduce the rate of progressionto BK viremia and BKVN.4. BK viremia could be damage to graft function. Therefore, we should reduce theimmunosuppressive drugs and other clinical intervention in time when BK viremia,espectially sustain viremia.5. Stimulation BKV specific cellular immune response in vitro is given priority towith CD4+T cell immune response after renal transplantation recipients. Frequency ofvirus cell specific immunity response was declined significantly with stimulating structuralproteins VP1, V2and VP3in virtro when Viremia and BKVN. 6. Structural protein VP1and VP3could be act the leading roleb in BKV specificimmune response, because frequency of virus cell specific immunity response in vitro withthese two kinds of structural protein were significantly higher than VP2, LT and st.7. BKV specific immune response was suppressed severly when the BKV infectionsatge progressed at viremia, if the body could not recover the antiviral capacity in time,sustain viremia condition developing to BKVN easily and kidney function could bedamaged and even graft loss.