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Control of human cytomegalovirus (HCMV) infection during the posttransplant period was investigated in 134 solid-organ transplant recipients by monitoring in parallel virologic and immunologic parameters for at least 1 year of follow-up. Virologic monitoring was achieved by determining HCMV DNAemia with real-time PCR, using the threshold of 300 000 DNA copies/mL blood as a cutoff for starting preemptive therapy. Immunologic monitoring included measurement of HCMV-specific CD4+ and CD8+ T cells by cytokine flow cytometry, using HCMV-infected dendritic cells as a stimulus. HCMV infection was diagnosed in 110 (82%) and required treatment in 49 (36%) patients. At 12 months after transplantation ‘protective’ immunity (≥0.4 CD4+ and CD8+ HCMV-specific T cells/μL blood) was achieved in 115/129 (89%) patients. During the entire study period, 122 patients reconstituting HCMV-specific CD4+ and CD8+ T-cell immunity at 60 days posttransplant onward were able to control HCMV infection, except for one patient who developed HCMV disease because of a rejection episode. Patients reconstituting HCMV-specific CD8+ only did not control HCMV infection. In conclusion, the presence of both HCMV-specific CD4+ and CD8+ T cells ≥ 0.4/μL blood appears to be protective against HCMV disease. This result does not apply to patients undergoing antirejection treatment, or reconstituting HCMV-specific CD8+ T cells only.
HCMV infection/disease still remains the major infectious viral complication in the posttransplant period for solid-organ transplant recipients (1). Antiviral drugs have been very efficient both in treating and preventing HCMV disease (2) by either the preemptive therapy or prophylactic approach (3–6). During the last decade it has been confirmed by several groups that the severity of HCMV infection inversely correlates with the development or restoration of efficient HCMV-specific T-cell immunity (7–8). In addition, it has been shown that long-term protection from HCMV reactivation is achieved through HCMV-specific T-cell immunity (9–12). However, long-term prospective studies addressing the possibility of tailoring HCMV prevention strategies based on determination of HCMV-specific T-cell responses in the posttransplant period are still lacking.
Furthermore, the use of different methodologies for the evaluation of the T-cell response has hampered the comprehensive evaluation of the cellular immune response. Apart from initial assays (13); more recent technologies have all been useful in providing some indications on immune protection from HCMV disease (14–16). In most studies, antigenic stimulation was provided by synthetic peptides (17) or infected cell lysates. While the immune response to single-protein (such as pp65 and IE-1) peptide mixtures was shown to be variable and inconsistent (18), the use of a pool of 34 peptides relevant to different viral proteins (stimulation of CD8+) and infected cell lysates (stimulation of CD4+) was found to provide satisfactory results (19). In order to overcome problems related to partial stimulation, we introduced, and widely employed (11, 12,18,19), a new methodology aimed at stimulating both CD4+ and CD8+ T cells with HCMV-infected autologous dendritic cells (DC) (20).
In 134 solid-organ transplant recipients, we conducted, in parallel, virologic monitoring of HCMV infection by real-time PCR (6,21) and immunological monitoring with the infected DC technique. Results show that levels of specific CD4+ and CD8+ T cells greater than 0.4/μL blood are highly predictive of protection from HCMV disease, in the absence of antirejection steroid treatment.
Patients and Methods
Subjects and study design
From March 2007 through February 2010, 134 patients, receiving a solid-organ transplant at the University Hospital, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy, were enrolled in a prospective observational study. Virologic and immunological monitoring of HCMV infection was performed for at least 1 year. The study was aimed at verifying whether previously established levels of HCMV-specific CD4+ and CD8+ T cells (11) were able to confer protection from HCMV disease. The 134 patients analyzed included 58 heart transplant recipients (HTR), 52 kidney transplant recipients (KTR) and 24 lung transplant recipients (LTR). Inclusion criteria included male and female subjects >18 years of age, and serological evidence of past HCMV infection in either the organ recipient or donor.
The immune response is considered protective when it can control infection in blood in at least 95% of cases. On the basis of a previous study (11), we chose levels ≥ 0.4 HCMV-specific CD4+ and CD8+ T cells/μL blood (in the absence of antirejection treatment) as the immunological cutoff. In this case, the proportion of patients reaching 300 000 HCMV DNA copies/μL blood (the cutoff currently used for initiating preemptive therapy) in the presence of ≥0.4 HCMV-specific CD4+ and CD8+ T cells/μL blood should be less than 5%. Assuming a study power ≥80% and adopting a binomial distribution model to calculate the 95% confidence interval of the ‘failure’ rate, the upper limit of this interval would be ≤5%, if no more than 3 out of 130 patients reach the blood cutoff for preemptive therapy after immune recovery. The study was approved by the local Ethics Committee (procedure no. P-20060029007 of February 19, 2007), and participants gave written informed consent. Patient characteristics and immunosuppressive drug regimens are reported in Table 1.
HCMV infection diagnosis, monitoring and treatment
The donor/recipient serostatus was determined by the enzyme-linked immunosorbent assay prior to transplantation. HCMV infection was defined as HCMV detection in blood or body tissues, whereas HCMV disease required, in addition to virus detection, the presence of clinical symptoms and/or organ function abnormalities (2). Management of HCMV infection followed the guidelines provided by the SIV-AMCLI (22) and The Transplantation Society (5). During the first 3 months after transplantation, HCMV infection was monitored by determining the presence of HCMV DNA in blood (DNAemia) by real-time PCR (21) once a week in the case of undetectable infection, and twice a week in the case of HCMV infection. Subsequently, clinical and virological monitoring was performed monthly until at least 1 year after transplantation. Local HCMV infection was diagnosed by a tissue biopsy. In LTR, HCMV was also routinely determined in bronchoalveolar lavage (BAL) fluid (12). Preemptive antiviral therapy for systemic HCMV infection was initiated when the cutoff of 300 000 DNA copies/mL blood (6,22) was reached. For LTR, preemptive therapy was initiated not only when the cutoff value for DNAemia was reached, but also when the DNA level in BAL exceeded 100 000 copies/mL (12,22). HCMV infection relapses were treated similarly. The same preemptive approach was used in the case of rejection episodes. Antiviral therapy routinely consisted of i.v. 5 mg/kg/bid ganciclovir (GCV) or 900 mg/bid valganciclovir (VGCV). When necessary, for toxicity problems or GCV-resistance, GCV was replaced with foscarnet (PFA) at the dosage of 90 mg/kg/bid.
HCMV-specific CD4+ and CD8+ T cells were measured with an in-house developed method based on the use of autologous, monocyte-derived, HCMV-infected immature DC, as previously reported (20). Briefly, following in vitro generation, DCs were infected with an endotheliotropic and leukotropic strain of HCMV (VR1814) (23). Infected DCs were then cocultured overnight with autologous PBMC at a ratio of 1:20 in the presence of brefeldin A. The frequency of HCMV-specific CD4+ and CD8+ T cells producing interferon γ (IFN-γ) was determined by CFC (14,20). Levels of HCMV-specific IFN-γ+ CD4+ and CD8+ T cells greater than 0.4 cells/μL blood (and 0.05% of either cell subset) were considered protective from HCMV disease (11). In addition, HCMV-specific T cells able to produce IL-2 in addition to IFN-γ were also measured by CFC (IFN-γ+/IL-2+ T cells). Immunological assays were performed monthly until day 180 after transplantation, then every 3 months until detection of HCMV-specific CD4+ and CD8+ T cells, or until at least 1 year of follow-up.
The immunosuppressive drug concentration in blood was routinely determined in all transplanted patients in order to maintain drug levels within the therapeutic range and avoid toxic effects. The antiviral drug concentration in blood was measured in the case of suspected drug ineffectiveness (i.e. stable HCMV DNA or increasing levels during therapy).
Curves relevant to restoration of specific CD4+ and CD8+ T-cell immunity were determined by the Kaplan–Meier method, while differences between curves were determined by the log-rank test. Differences between proportions were determined by Fisher's exact test and differences between medians by the Mann–Whitney U-test.
HCMV infection and antiviral treatment
HCMV infection was diagnosed in 110/134 (82%) patients. In detail, HCMV infection was detected in 48/58 (83%) HTR, 42/52 (81%) KTR and 20/24 (83%) LTR.
Antiviral treatment was given to 49/134 (36%) patients. In detail, antiviral treatment was given to 14/58 (24%) HTR, 17/52 (33%) KTR and 18/24 (75%) LTR. Among the 49 patients treated, 39 were treated preemptively after reaching the systemic DNAemia cutoff (n = 26) or, in LTR, the DNA cutoff in BAL (n = 11) or both cutoffs (n = 2). In addition, eight patients had a symptomatic organ localization before reaching (n = 3) or concomitantly with the (n = 5) DNAemia cutoff, and two patients had a symptomatic systemic syndrome (fever, leukopenia, thrombocytopenia, rise in liver enzymes) concomitantly with the DNAemia cutoff. Median HCMV DNA viral load levels were 2700 (range < 100–204 000) DNA copies/mL blood for the 77 patients spontaneously resolving the infection, 322 200 (<100–3 965 000) for the 39 patients treated with preemptive therapy and 474 450 (19 600–1 306 000) for the 10 patients with HCMV disease (Figure 1). GCV or VGCV therapy was able to clear the infection in all cases after a median treatment duration of 28 days (range 13–192). However, in one patient, GCV had to be replaced by PFA due to the low plasma GCV concentration.
The median duration of follow-up was 799 days (range 82–1410). All but five patients, who died within the first year after transplantation, have been followed up for at least 1 year. Four additional patients died more than 1 year after transplantation.
Recovery of HCMV-specific T-cell immunity
As previously defined, immune-protection from HCMV disease in solid-organ transplant recipients is supported by the simultaneous presence of ≥0.4 HCMV-specific IFN-γ+CD4+ and IFN-γ+CD8+ T cells/μL whole blood (11). As shown in Figure 2A, protective immunity was reached within 3 months in 72/132 (55%) patients, within 6 months in 106/131 (81%) patients and within 12 months in 115/129 (89%) surviving patients. In addition, HCMV-specific CD4+ T-cell reconstitution was reached significantly earlier (p = 0.015) in patients with baseline-specific CD4+ T-cell count greater than 0.4 cells/μL compared to patients with a CD4+ T-cell count lower than 0.4 cells/μL (Figure 2B). The HCMV-specific T-cell reconstitution occurred significantly (p < 0.001) earlier in the heart compared to lung and kidney recipients (Figure 2C).
The cumulative incidence of reconstituted HCMV-specific CD4+ and CD8+ T-cell responses is reported in Figure 3A, where CD8+ T cell reconstitution appears to precede CD4+ T-cell reconstitution. The difference between the two curves was found to be significant (log-rank test, p = 0.001), with a median time of 57 days for CD8+ T-cell reconstitution versus 87 days for CD4+ T-cell reconstitution. An even greater delay was observed for reconstitution of both IFN-γ+/IL-2+ CD8+ (132 days) and CD4+ (155 days) T-cells.
Primary versus reactivated HCMV infection: T-cell response and HCMV clearance
On the whole, 11 patients with primary HCMV infection and 117 patients with reactivated infection were analyzed. Of these (Table 2), 9/11 patients with primary infection (82%) and 40/117 (34%) with reactivated infection (p = 0.003) required antiviral treatment. Similarly, 3/11 (27%) patients with primary infection, and 7/117 (6%) with reactivated infection (p = 0.041) developed HCMV disease. Appearance of HCMV-specific IFN-γ+CD4+ T cells was significantly delayed in primary versus reactivated infections (p = 0.044). The same trend was observed for HCMV-specific IFN-γ+/IL-2+CD4+ T cells (p = 0.012). No significant difference was found between the two groups for IFN-γ+CD8+ and IFN-γ+/IL-2+CD8+ T cells (Figure 3B and Table 2). Similarly, HCMV clearance was not significantly different between the two groups of patients (Figure 3C and Table 2). However, the viral load was significantly (p = 0.002) higher in primary compared to reactivated infections (Table 2).
Table 2. Comparison of immunologic, virologic and clinical parameters in solid-organ transplanted patients with primary (n = 11) or reactivated (n = 117) HCMV infection
Primary infection (n = 11)
Reactivated infection (n = 117)
NA, not applicable.
3Fisher's exact test.
HCMV-specific IFN-γ+/ CD4+ (median time, days)1
HCMV-specific IFN-γ+/ CD8+ (median time, days)1
HCMV-specific IFN-γ+/IL-2+CD4+ (median time, days)1
HCMV-specific IFN-γ+/IL-2+CD8+ (median time, days)1
Peak HCMV DNAemia level (median, range)2
398 700 (32 000–1 008 000)
30 600 (400–3 965 000)
HCMV clearance (median time, days)1
Treated patients (%)3
Patients with HCMV disease (%)3
Control of HCMV infection by the HCMV-specific T-cell response
In 4/35 patients, the T-cell response detected during the first month after transplantation was lost during the second month. These four patients developed a systemic HCMV infection requiring preemptive therapy prior to final T-cell response restoration which was maintained thereafter. From the end of the second month, the HCMV-specific T-cell response was sustained and no patient reached the HCMV DNA cutoff in blood (except in the case of antirejection treatment). Thus, the end of the second month after transplantation was selected as the first reliable time point for immune recovery assessment.
Of the 134 patients examined in this study (117 HCMV-seropositive and 17 HCMV-seronegative), 128 reconstituted (n = 117) or developed (n = 11) a T-cell response during follow-up (Figure 4). Of the remaining six HCMV-seronegative patients, five did not develop primary infection and one died of multiorgan failure 86 days after transplantation before developing immunity. Overall, during the entire study period, 122/128 (95%) patients recovered both HCMV-specific CD4+ and CD8+ T cells (115 patients within 1 year, and seven patients between 400 and 600 days after transplantation), while six patients showed only a CD8+ T-cell response. Overall, 121 patients spontaneously controlled blood infection. Of these, five patients (4%) were treated because of HCMV DNA levels in BAL above the cutoff in the absence of signs or symptoms of pneumonia (two concomitantly with acute rejection). The only patient unable to control HCMV infection received high-dosage steroid therapy for acute rejection, and developed an HCMV systemic syndrome in association with pneumonia, which was successfully treated.
In addition, among the 47 patients in whom HCMV-specific CD8+ appeared before CD4+ T cells during the follow-up, as many as 11 (23.4%) developed either a systemic HCMV infection (n = 5), or lung infection (one symptomatic) (n = 6), both requiring antiviral treatment.
Immune recovery and HCMV infection according to the immunosuppressive drug regimen
As reported in Figure 5, HCMV-specific CD4+ (A) and CD8+ (B) T cells were recovered earlier in patients receiving everolimus (RAD, rapamycin derivative) compared to mycophenolate mofetil (MMF) containing regimens (the log-rank test, p = 0.01). In addition, the incidence of HCMV infection (Figure 5C) was significantly lower (p < 0.01) in patients receiving RAD (in association with CSA and steroids) compared to patients receiving CSA+MMF+steroids or TAC+MMF+steroids. Furthermore, all patients receiving RAD spontaneously resolved the infection.
Immunosuppression was tapered in three patients prior to reaching the DNAemia cutoff in an attempt to control HCMV infection without antiviral drug administration. Two KTR with no immune reconstitution reached the cutoff, and antiviral chemotherapy was started. In a HTR with immune reconstitution, HCMV infection resolved spontaneously.
HCMV-specific T-cell reconstitution and acute rejection
Overall, 29/134 (21.6%), patients experienced 35 episodes of acute rejection at a median time of 96 (range 3–257) days after transplantation. Five episodes resolved spontaneously. Of the remaining 30 episodes, 15 were treated with 3-day administration of steroid bolus, 9 with increased steroid dosage, 4 with ATG, 1 with increased CSA dosage and 1 with a shift from sirolimus to TAC. HCMV infection was observed concomitantly or after antirejection treatment in 15/30 (50%) episodes. In detail, in nine cases HCMV infection resolved spontaneously. In the remaining six cases, the cutoff for preemptive therapy was reached either in blood (n = 2) or in BAL (n = 2), while HCMV disease was diagnosed in two patients.
Measurement of HCMV-specific T-cell responses before and after (median interval 29, range 3–72, days) treatment for rejection was available for 19 episodes relevant to 17 patients. Of these 4/5 (80%) patients with only a CD8+ T-cell response, prior to antirejection treatment, developed an HCMV infection, whereas only 6/14 (43%) patients, displaying both CD4+ and CD8+ T-cell responses before treatment, developed an HCMV infection. However, this difference was not significant.
Results of this study point to the following conclusions: (i) HCMV-specific immune responses can efficiently control HCMV infection; (ii) the HCMV-specific CD4+ and CD8+ T-cell cutoffs previously established (≥0.4 T cells/μL blood) have been confirmed prospectively as predictive of protection from HCMV disease; (iii) HCMV-specific T-cell responses during the first 2 months after transplantation may run an irregular course, with some patients losing their response after initial recovery; (iv) in the absence of HCMV-specific CD4+ T cells, CD8+ T cells do not appear to be able to consistently control HCMV infection; (v) patients receiving RAD recover HCMV-specific T-cell responses earlier than patients receiving MMF, thus undergoing a significantly lower rate of HCMV infection episodes; (vi) during organ rejection episodes, reconstituted T-cell immunity may be lost either phenotypically or functionally, thus leading to new episodes of HCMV infection.
Cutoff levels for preemptive therapy have been selected based on different criteria in different transplantation centers using preemptive therapy instead of prophylaxis (25,26). In our hospital, the cutoff of 300 000 DNA copies/mL blood was established by prospectively following a large number of solid organ transplant recipients during the first year after transplantation (21,22,24) and has been consistently used since 2004. This cutoff has been questioned by some authors (25,26) as being too high. Although lower cutoff levels are considered sufficient to prime the immune response, our higher cutoff level may be more efficient in eliciting the HCMV-specific immune response. We have clinically validated our cutoff in a large series of patients (6), and in a multicenter quality control study for HCMV DNAemia quantification (27). Results of this study further confirm its validity and support the possibility of applying the same prevention strategy to different types of transplant and to both seropositive and seronegative recipients, although the latter showed (as expected) delayed immune protection and a more frequent need for preemptive therapy. However, in spite of the significant difference in peak viral load between primary and reactivated infections, time to virus clearance did not differ. This is likely due to the antiviral treatment of most primary infections (82%) versus spontaneous resolution of most (66%) reactivated infections.
In this study, viral load levels below the established cutoff were consistently controlled by the T-cell immune response (HCMV-specific CD4+ and CD8+ T cells ≥ 0.4/μL). In fact, in 95% of transplanted patients evaluated, the presence of a minimum level of 0.4 cells/μL blood for HCMV-specific CD4+ and CD8+ T cells assured either the control or abortive outcome of HCMV infection and prevention of HCMV disease. Only when patients faced an episode of rejection, requiring adoption of high-dosage steroid therapy or an increase in the immunosuppressive regimen, was T-cell protection lost for a variable period of time. The phenotypic and functional T-cell defects induced by steroid therapy remain to be identified. In addition, the protection level conferred solely by the HCMV-specific CD8+ T-cell response remains to be investigated.
The only time period in which apparently protective levels of T-cell immunity were possibly lost was during the second month after transplantation. After that time, in the absence of antirejection treatment, the recovered T-cell response was able to control systemic HCMV infection in all patients examined, thus verifying the primary objective of this study. This observation indicates that the end of the second month is the time point when, in the absence of rejection episodes, reconstituted T-cell immunity efficiently persists over the following months. Thus, from this time point on, it should be possible to discontinue virologic monitoring in blood when HCMV-specific CD4+ and CD8+ T-cell responses are detected above the established cutoffs, as already proposed for hematopoietic stem cell transplant recipients (28).
In a previous study, it was shown that HCMV-specific CD4+ T cells play a major role in long-term protection from HCMV infection (11). In the past, a dominance of HCMV-specific CD8+ T cells was reported in the early response to primary HCMV infection (29) in contrast to a relatively predominant HCMV-specific CD4+ T-cell response in long-term protection in persistent or latent infections (9,30). In addition, it was shown that in symptomatic infections the HCMV-specific CD8+ T-cell response precedes the CD4+ response and does not confer protection, whereas in asymptomatic infections of renal transplanted patients with primary HCMV infection the HCMV-specific CD4+ precedes the HCMV-specific CD8+ T-cell response (31,32). Results of this study indicate and confirm that only in the presence of an HCMV-specific CD4+ T-cell response is long-term protection conferred. Preliminary observations of this study indicate that protection conferred by HCMV-specific CD4+ T cells somewhat correlates with the neutralizing antibody response. In fact, in a subgroup of 11 patients examined, a trend toward a higher neutralizing antibody titer (p = 0.07) was observed in patients not reaching versus those reaching the cutoff for preemptive therapy, as well as in the presence versus absence of HCMV disease (data not reported).
The level of functional activity inhibition exerted by different immunosuppressive drugs is markedly different. In our study, the previously reported reduction in the rate of HCMV infections in KTR and HTR patients receiving RAD was confirmed with respect to transplanted patients receiving immunosuppressive regimens not containing RAD (6,33–35). In addition, earlier induction of the T-cell response was detected in our study in patients receiving RAD.
While systemic HCMV infection/disease may efficiently be prevented by the HCMV-specific T-cell response, at least in the absence of additional immunosuppressive drug effects, it appears much more difficult to promptly detect local organ infection, particularly in the absence of symptoms or viral load in blood (36,37). In this respect, the LTR setting, where periodical BAL sampling is performed posttransplant, is peculiar. HCMV in BAL is currently considered a correlate of HCMV lung infection, and replicate BAL sampling during rejection surveillance may represent a suitable material for early HCMV detection in the lung. However, the true prognostic significance of HCMV detection in BAL remains to be defined (12,36,38,39). In a previous study correlating viral load in BAL with virus detection in lung biopsies, we found that levels >1×105 HCMV DNA copies/mL BAL were detected in 100% of LTR with HCMV pneumonia (lung infection + inflammatory infiltrates) (12), in 25–30% of patients with HCMV infection in lungs and no inflammatory infiltrates, and only 3% of LTR with no signs of lung infection. In the same study, we selected a cutoff of 100 000 DNA copies/mL BAL for initiating preemptive therapy. The same guidelines were adopted in this study, and no HCMV infectious complications were observed in transplanted lungs. However, a clinically validated cutoff for HCMV DNA in BAL to guide preemptive therapy in LTR remains to be identified in a randomized prospective study.
In conclusion, HCMV-specific T-cell response monitoring can be coupled with virological monitoring to efficiently guide HCMV disease prevention by preemptive therapy. For this purpose, if our DC-based assay, which takes 7 days to complete, is cumbersome and time consuming, and may not be applied at other transplantation centers, more rapid immunological assays providing results within 24 h may be satisfactorily performed (19).
We thank the technical staff of the Virology Section of the Struttura Complessa of Virology and Microbiology for performing the viral assays. We acknowledge the careful secretarial and informatic support of Daniela Sartori and thank Laurene Kelly for English revision. We are indebted to Piero De Stefano for helpful discussion. This work was partially supported by grants from the Fondazione Carlo Denegri, Torino, and by the Ministero della Salute, Ricerca Corrente IRCCS Policlinico San Matteo (grants 80221 and 80425).
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.