Microbiol Immunol 2014; 58: 72–75 doi: 10.1111/1348-0421.12112
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The presence of antibodies against the AD2 epitope of cytomegalovirus glycoprotein B is associated with acute rejection after renal transplantation Kei Ishibashi1,2, Tadahiko Tokumoto3, Hiroki Shirakawa3, Toshiki Oguro1, Tomohiko Yanagida1, Norio Takahashi1, Masanori Nomiya1, Nobuhiro Haga1, Ken Aikawa1, Kazunari Tanabe3, Naoki Inoue4, Yoshiyuki Kojima1 and Tatsuo Suzutani2 1
Department of Urology, 2Department of Microbiology, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960‐1295, 3Department of Urology, Tokyo Women's Medical University, 8‐1 Kawadatyo, Shinjuku‐ku, Tokyo 162‐8666 and 4Department of Virology I, National Institute of Infectious Diseases, 1‐23‐1, Toyama Shinjuku‐ku, Tokyo 162‐8640, Japan
ABSTRACT The aim of this study was to evaluate the association between antibodies against cytomegalovirus (CMV) glycoprotein B (gB) and acute rejection after transplantation. Seventy‐seven consecutive renal transplant recipients in a D þ /Rþ setting were studied. Biopsy‐proven rejection occurred in 35% of the recipients. Among these recipients, 85% had antibodies against CMV gB. The rate of acute rejection was significantly higher in recipients with antibodies against gB than in those without them. Antibodies against gB can be a useful predictor of acute rejection in renal transplant recipients in a D þ /Rþ setting. Key words
acute rejection, cytomegalovirus, glycoprotein B, renal transplantation.
Renal transplantation is a most valuable treatment for patients with end‐stage renal disease, offering a long‐term survival benefit compared with patients on dialysis (1). However, acute rejection episodes are an important risk factor for functional deterioration of solid‐organ transplants (2). Although novel immunosuppressive regimens have reduced graft loss, susceptibility to infections has increased. Viral replication after transplantation may contribute to reduced graft function and survival through the associated inflammation and cytokine release (3). Uncontrolled replication of viruses such as adenovirus, CMV, polyomavirus BK, John Cunningham virus, parvovirus B19 and human herpes virus‐6 and ‐7 triggers direct and/or indirect effect in transplant recipients (4). Among these viruses, CMV is the most important pathogen affecting kidney allograft recipients. When CMV is reactivated under immunosuppressive condi-
tions, it has both direct effects, such as development of CMV disease, and indirect effects on transplantation, including increased incidence of allograft rejection (5). The role of CMV infection in acute rejection after renal transplantation remains controversial; several studies have suggested that it can lead to allograft rejection (6, 7). Because investigation of strategies for preventing CMV replication and acute rejection is of ongoing interest (8), we have concentrated on this matter in our series of our studies. Cytomegalovirus, a member of the herpesvirus family, has a large genome which encodes over 65 unique glycoproteins (9). It is well known that some of the glycoproteins encoded by CMV induce strong immune responses, as do other viral components. Among the glycoproteins gB, one of the most abundant envelope components, is essential for viral replication
Correspondence Kei Ishibashi, Department of Urology, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960‐1295, Japan. Tel: þ81 24 547 1316; fax: þ81 24 548 3393; email:
[email protected] Received 21 August 2013; revised 30 September 2013; accepted 22 October 2013. List of Abbreviations: a.a., amino acid; AD2, antigen domain 2; CMV, cytomegalovirus; Dþ/Rþ, both donor and recipient positive for CMV; gB, glycoprotein B; gH, glycoprotein H; gH‐m, gH‐type‐matched; GST, glutathione S‐transferase.
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CMV gB antibodies and acute rejection
and considered one of the major target molecules for neutralizing antibodies as well as for cellular immune response (10). Three linear antibody‐binding sites have been described: it is well known that the AD2 site I epitope of gB is conserved in CMV isolates and is the major epitope for neutralization (9, 11, 12). The antibody‐binding site on AD2 is located between a.a. 28 and 84 of gB (9, 11). gB is also a target for CMV‐ specific T‐cell immunity. Although little is known about any association between gB AD2 and CMV‐specific T‐cells, Elkington et al. isolated CD4þ cytotoxic T lymphocytes (13), which recognize epitopes from CMV gB in association with HLA‐DR7 and DR11 antigens. In addition to gB, gH has been used to identify preexisting strain‐specific antibodies (14, 15). Previously, we found that reinfection of seropositive recipients with a different type of CMV is also associated with acute rejection and CMV disease in renal transplant patients (15). A study which reevaluated the previous study has also indicated that the absence of antibodies against gB in transplantation recipients is a good indicator of CMV disease (16). In this study, we investigated whether, in addition to CMV disease, antibodies against gB AD2 contribute to prediction of acute rejection in renal transplantation in D þ Rþ setting, irrespective of gH serological matching. This study investigated 77 CMV seropositive renal transplant recipients whose donors were also CMV seropositive (D þ /Rþ setting) and in whom antibodies against amino‐terminal regions of CMV‐gH had been detected; these recipients were enrolled at Fukushima Medical University and Tokyo Women's Medical University and have been described previously (15). All study recipients had received hemodialysis treatment before transplantation and had received living‐related renal transplants. This study was approved by the Institutional Ethics Committee and written informed consent was obtained from all subjects. All serum specimens were obtained before transplantation. To detect antibody against CMV gB AD2 site I, which is located between a.a. positions 68 and 77 of gB of AD169 strain, GST protein fused with the AD2 site I was expressed and purified as described previously (17). DNA cassette encoding the conserved epitope in CMVAD2 site I was cloned into the expression vector pGEX‐5X (Amersham Bioscience [now GE Healthcare], Piscataway, NJ, USA). GST fusion proteins containing the gH epitopes from the AD169 and Towne strain were used to detect CMV gH type‐specific antibodies as previously reported (15). OD values specific to each antigen were obtained by subtracting the OD values for GST as described previously (15). An arbitrary cutoff for ELISA (OD ¼ 0.25) was defined as the mean plus two standard deviations of OD values of a panel of healthy CMV © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Table 1. Baseline characteristics CMV gB serostatus No. of pairings Age (years; mean SD) HLA mismatch (A, B, DR) No. of immunosuppression (%) Tacrolimus‐based regimen Cyclosporin‐based regimen
gB (þ)
gB ()
52 41.4 12.6 2.9 1.3
25 42.9 14.8 2.6 1.1
50 (96) 2 (4)
24 (96) 1 (4)
seronegative volunteers (15). Detection of strain‐specific gH‐antibodies in the recipients' serum samples, which matched those of their donors, was considered gH‐m antibody positivity. The basic characteristics of the renal transplant recipients are summarized in Table 1. Fifty‐two of the 77 recipients had antibodies against gB. There were no differences between patients with and without gB antibodies in other relevant variables, namely age, sex, number of HLA mismatches and immunosuppression protocols. The transplant recipients were followed up for 6 months after transplantation. Rejection was suspected when serum creatinine concentrations increased more than 25% above the basal level in the absence of urinary tract obstruction or renal graft artery stenosis, as described previously (15). The first rejection episode was confirmed histologically by biopsying the grafts. Preemptive therapy was employed when CMV infection and/or CMV end‐organ disease were diagnosed, as described previously (15). Using StatView 5.0, Fisher's exact test was used to evaluate the rate of acute rejection in different gB serostatus groups. Statistical significance was set at P < 0.05. The incidence of biopsy‐proven acute rejection was calculated using the Kaplan–Mayer method, and comparisons were carried out by the log‐rank test using SPSS. Subsequent to their entry into the study, 27/77 recipients (35%) in a D þ /Rþ setting experienced biopsy‐proven rejection during the 6 months after transplantation. Among these 27 D þ /Rþ patients with rejection, 23 (85%) had antibodies against CMV gB. The incidences of acute rejection among recipients with (gBþ) and without (gB) antibodies against gB AD2 were 44% and 16%, respectively. The rate of acute rejection was significantly higher in gBþ recipients than in gB recipients (Table 2). Figure 1 shows Kaplan–Meier curves for the cumulative probability of freedom from biopsy‐proven acute rejection. There were significant differences between the gBþ group and the gB group according to the log‐rank test (P ¼ 0.025). Our previous study, which employed multivariate logistic regression analysis to identify factors associated 73
K. Ishibashi et al.
Table 2. Comparison of incidence of acute rejection according to antibody response against CMB gB AD2 CMV gB serostatus
gB (þ)
gB ()
P
No. of recipients No. of recipients with acute rejection (%)
52 23 (44)
25 4 (16)
0.021
Fig. 1. Kaplan–Meier curves of the cumulative probability of freedom from biopsy‐proven acute rejection. The incidence of acute rejection in the gB þ group is significantly higher than that in the gB group (P ¼ 0.025).
with acute rejection, including age, sex, number of HLA mismatch, immunosuppression protocols, demonstrated that reinfection of seropositive recipients with a different gH‐serotype of CMV is associated with adverse outcomes in renal transplant recipients (15). On the other hand, in the present study, there was no significant difference in the gB antibody‐positive rate between gH‐mþ and gH‐ m recipients with acute rejection (Table 3), suggesting that presence of antibodies against gB is a risk factor irrespective of gH serological matching. Many studies have reported a relationship between CMVand allograft rejection in renal transplant recipients. Previously, we reported that mismatch of gH antibody types between donors and recipients of renal transplan-
Table 3. gB AD2 seroprevalence according to gH‐type matching in the 27 recipients with acute rejection gH type match
No. of recipients with rejection
(þ)
()
P
10
17
ns
No. of gB (þ) recipients with acute rejection (%) 9 (90)
74
14 (82)
tation in a D þ /Rþ setting, which probably indicates reinfection with a strain different from the original CMV strain, is associated with acute rejection after transplantation (15). In this study, we revisited the risk of acute rejection in the same cases and found that 23 of the 27 recipients who experienced biopsy‐proven acute rejection during the 6 months follow up after transplantation had antibodies against CMV gB AD2, indicating that the presence of antibodies against the gB AD2 may be a good predictor of rejection in recipients in a D þ /Rþ setting. About 30–70% of CMV positive subjects have antibodies against gB AD2 (9, 17), which is one of the major epitopes for neutralizing antibodies (9, 11). That the prevalence of antibodies against gB is similar in gH‐ matched and ‐mismatched recipients with acute rejection, suggests that the presence of gB antibodies is a risk factor, independent of mismatch of gH serotypes. Because of the limited number of recipients with acute rejection, further study of a larger patient group is required to confirm this finding. Nevertheless, we postulate that immune responses against CMV gB, which our ELISA system detected, may be associated with acute rejection. Although CMV‐specific cellular immunity provides protection by limiting CMV reactivation and replication, it is plausible that acute rejection is a consequence of strong cell‐mediated responses against ongoing CMVactivity. Because gB is one of the significant targets for CMV‐specific CD8þ and CD4þ T‐cell immunity (10, 18), it would be interesting to ascertain whether CMV‐specific T‐cell activity against CMV‐gB correlates with the outcome of our ELISA findings concerning gB AD2. Endogenous CMV‐gB is presented efficiently by MHC Class II molecules of endothelial, epithelial and glial cells and can promote CD4þ T‐cell recognition (19). In conclusion, this study, which reevaluated a previous study, indicates that the presence of antibodies against gB in transplantation recipients may be a good indicator of possible acute rejection. Further study are needed to evaluate the association between antibody responses against gB and cellular immune responses in renal transplant recipients.
ACKNOWLEDGMENTS We thank all the subjects who participated in this study. This work was supported by a Grant‐in‐Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 16591609).
DISCLOSURE No authors have any conflicts of interest to declare. © 2013 The Societies and Wiley Publishing Asia Pty Ltd
CMV gB antibodies and acute rejection
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