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Adenovirus infection in children after allogeneic stem cell transplantation: diagnosis, treatment and immunity

Summary:

Human adenoviruses (HAdV) are a frequent cause of potentially fatal infections in patients after allogeneic stem cell transplantation, especially in children. Monitoring of serum/plasma by real-time quantitative PCR is a sensitive tool for the recognition of patients at risk of a potentially fatal infection and for the evaluation of the efficacy of treatment. Data from a retrospective study and from a prospective study demonstrate that recovery of immunity after transplantation is essential for the elimination of HAdV infection. The feasibility of several approaches for the manipulation of immunity in the immunocompromised host to prevent a fatal course of the infection is discussed.

Main

Human adenoviruses (HAdV) are increasingly recognized as pathogens causing life-threatening infections in patients after allogeneic stem cell transplantation (SCT) (reviewed in Walls et al1 and Kojaoghlanian et al2). Currently, 51 serotypes of HAdV are known which are grouped into six species (A–F). Serotypes from species A, B and C are commonly reported as the infectious HAdV type in SCT recipients. The incidence of HAdV infections is higher in paediatric than in adult SCT recipients, which might be related to the high exposure to HAdV at young age, while HAdV-specific immunity has still to be mounted.3, 4

In a retrospective study of a cohort of 328 children consecutively transplanted between 1985 and 1999 in our institute with a graft from an allogeneic donor, 37 cases with HAdV infection were recognized (van Tol et al, paper submitted). Of 37 patients, 17 developed HAdV disease that had a fatal course in seven of them. Three patients were treated with antiviral drugs (ribavirin or cidofovir), but only in the last week before decease. From this study, donor type (ORD/MUD), strong modulation of the graft, use of melphalan in the conditioning and recipient age below 5 years were identified in a multivariate statistical analysis as risk factors for the occurrence of HAdV infection. Recipients aged below 5 years who received a rigorously (>2 log) depleted graft from an ORD/MUD donor were defined as a high-risk group with an 84% actuarial incidence of HAdV infection at 6 months after SCT. Failure of engraftment or rejection of the graft, conditions associated with an extension of the period of impaired immunity, was a significant risk factor of the occurrence of HAdV disease in univariate statistical analysis. Immunosuppression consisting of ATG or Campath as part of the conditioning regimen was recognized as risk factor for HAdV infection in a prospective study of 48 children transplanted between 2001 and 2003 at our institute.5

In paediatric SCT recipients transplanted with a graft from an adult haploidentical family (ORD) or matched-unrelated donor (MUD), reactivation of endogenous HAdV is likely to occur. In this setting, modulation of the graft by depletion of T (and B) cells or enrichment of CD34+ progenitor cells and immunosuppression using polyclonal (ATG), monoclonal (Campath) antibodies or ciclosporin A are regularly applied. All these conditions have an impact on immune reconstitution and extend the period of impairment of adaptive immunity after SCT. Here, we summarize recent findings and current insights with respect to monitoring and treatment of HAdV infections. Based on the relationship between the occurrence and course of infection and immune recovery post SCT, options for immunotherapy are discussed.

Monitoring the occurrence and course of HAdV infection

Conventional monitoring of HAdV infection is usually performed by culture of faeces and urine samples and throat swabs. Recently, detection of HAdV DNA in plasma by real-time quantitative PCR has been introduced. In the retrospective study, serum samples of culture positive patients were analysed by semiquantitative PCR in addition to conventional weekly cultures. From this study, the following conclusions could be drawn: (a) spreading of HAdV over multiple sites only showed a trend to correlate with the development of HAdV disease (illustrated by Lankester et al6); (b) the duration of the infectious episode was not predictive of the course of the infection, for example, 40% of children with HAdV infection in the absence of clinical symptoms and only 50% of children with HAdV disease showed persistence of the virus. Persistence was defined by the presence of positive cultures for a period exceeding 14 days; (c) clinical symptoms other than diarrhoea became apparent rather late during the infectious episode; (d) a relatively high DNA load in serum turned out to be a sensitive and specific marker of a fatal course of the infection.7

In the prospective study, plasma samples were weekly obtained and analysed by real-time quantitative PCR (RQ-PCR). When infection progressed to viraemia, defined as the presence of an HAdV DNA load of 1000 copies/ml in two consecutive samples, the time point of first detection of DNA in plasma is usually very close to that of the first positive culture.5 Importantly, in most patients, a time window of several weeks was observed between the first detection of a significant HAdV DNA load in plasma and the terminal stage of HAdV disease, offering opportunity for intervention. Recently, Lion et al8 reported that detection of HAdV DNA in whole blood samples is associated with disseminated HAdV disease, which eventually may have a fatal course. Therefore, it can be concluded that monitoring of serum/plasma or whole blood by RQ-PCR is a sensitive tool for the recognition of patients at risk of a potentially fatal, disseminated HAdV infection.

Treatment of HAdV infection using antiviral drugs

There are numerous case reports and a few cohort studies on the use of the antiviral drugs ribavirin or cidofovir in the treatment of HAdV infection in SCT recipients.9, 10, 11 Failures and successes are reported for both drugs. A general conclusion from these studies was that rapid initiation of treatment after diagnosis of the infection is important. However, the value of most of these studies is limited because data on immune recovery in the patients have usually not been analysed simultaneously. For example, Miyamura et al12 have reported success of ribavirin treatment in recipients of a graft from an HLA-identical sibling donor in contrast to failure of treatment in recipients of a graft from an MUD. These findings could be explained by differences in the kinetics of immune reconstitution after SCT in these two patient groups. Ribavirin has been reported not to be effective in children with HAdV viraemia and without immune recovery.13

Cidofovir has been suggested to be effective in a report in which the immunocompetence of the patients was taken into account.10 However, prolonged treatment in immunocompromised patients is required, which could induce nephrotoxicity. It will be of interest to further explore the efficacy of an adapted treatment schedule of cidofovir in which a lower concentration of the drug is given at an increased frequency (3 × 1 mg/kg instead of 1 × 5 mg/kg weekly) aiming at a reduction of nephrotoxicity.11 Combined therapy with ribavirin and cidofovir has recently been claimed to have some synergistic effect compared to ribavirin alone in patients with HAdV infection of the respiratory and/or the gastrointestinal tract. Unfortunately, also in this study data on immunocompetence are lacking.14

In conclusion, in the majority of studies data on in vivo efficacy of antiviral drugs are interpreted without knowledge of the immunocompetence of the patients, while immune recovery appears to be a crucial risk factor for the occurrence and course of HAdV infection. Prospective studies on drug efficacy are required in which the course of HAdV infection is monitored by regularly performing cultures from different sites and RQ-PCR of plasma or blood in combination with proper investigation of the immune status of the patient. Ideally, investigation of the pharmacokinetics of the drugs should be included, especially because these data are virtually lacking in children. In addition, recent data of Morfin et al15 on in vitro drug susceptibility of clinical HAdV isolates show that HAdV strains from all species are sensitive to cidofovir, whereas only strains from species C are sensitive to ribavirin.

Immunity and HAdV infection

In comparison with other viruses such as Epstein–Barr virus (EBV) and cytomegalovirus (CMV), knowledge of the interaction between HAdV and the immune system is limited. Most likely, T cells as well as NK cells may play a role in recognition and killing of HAdV-infected target cells, while cytokines and neutralizing antibodies may limit the spread of the infection. In contrast to EBV and CMV, HAdV-derived epitopes recognized by T cells are only recently coming to be identified.16, 17 Furthermore, HadV-infected cells may escape from recognition by the immune system through several mechanisms.18

Evidence has been obtained that recovery of immunity after SCT is essential for the elimination of HAdV infection. The first indication was a case report in which a severe HAdV infection resolved in a patient after infusion of donor lymphocytes (DLI).19 HAdV secretion disappeared in four SCT recipients, who were treated for a leukaemia relapse after SCT either by tapering of ciclosporin A or by DLI infusion.20 An additional study in a larger cohort of patients showed a strong association between immune reconstitution and elimination of HAdV infection.21

Data obtained in ORD/MUD SCT recipients from our retrospective study indicated that patients with a low T-cell count (either CD4+ or CD8+ T cells) after SCT are at risk of developing an HAdV infection. In our prospective study,5 children with viraemia only survived the infection if lymphocyte numbers increased to above 0.3 × 109/l in the period of 4 weeks following the first detection of a positive RQ-PCR. An increase of lymphocyte numbers was clearly associated with a decrease of the plasma DNA load. Appearance of virus-specific CD4+ T cells in peripheral blood and a rise of neutralizing antibody titres specific for the HAdV serotype infecting the patient could be demonstrated after SCT. The relative contribution of neutralizing antibodies and specific T cells to the clearance of HAdV infection remains to be further investigated.

Immunotherapy for HAdV infection

As pointed out in the previous section, manipulation of immunocompetence in SCT recipients either by tapering of immunosuppression or infusion of DLI is an option for the pre-emptive treatment of patients at risk for development of HAdV viraemia. However, for obvious reasons, this approach will only be feasible in cases without acute GvHD or being not at risk to develop this complication. Furthermore, tapering of immunosuppression alone will only be effective in cases with some signs of immune reconstitution. In our opinion, initiation of immunotherapy could be considered in cases fulfilling the following conditions: (a) an HAdV DNA plasma load of 1000 copies/ml, (b) an increase of the load in a second sample taken within 1 week and (c) lymphocyte counts below 0.3 × 109/l.

In order to lower the risk of inducing acute GvHD by the infusion of unseparated DLI, selective depletion of alloreactive T cells is an attractive option. Using this approach, T cells recognizing infectious pathogens, for example, EBV, CMV and HAdV, will remain present in the T-cell population to be infused into the patient. Benefits of this strategy have been described, but occurrence of acute GvHD could not be avoided in all patients.22

Another approach, similar as has been described for EBV and CMV, would be to generate HAdV-specific T-cell lines from donor PBMC, thereby diluting out alloreactive T cells. In practice, T-cell cultures would have to be initiated before the HAdV serotype actually infecting the patient has been identified. Since HAdV-specific T cells generated against HAdV serotype 5 are crossreactive to HAdV serotypes from the same or even other species, this approach may be feasible.23 Recent work from our group has shown that HAdV-specific T cells, devoid of alloreactivity, can be generated using methylene blue-inactivated whole virus.23 To circumvent the preparation of GMP-compatible virus batches, a number of conserved peptides derived from HAdV 5 hexon has been identified that are recognized by T cells from the majority of adult blood bank donors. T cells expanded using these peptides recognize target cells infected with HAdV 5 or other HAdV types.24

Conclusions

In our experience, monitoring of patients with HAdV-specific RQ-PCR performed on plasma samples is an indispensable tool for the timely diagnosis of viraemia, for the instalment of pre-emptive therapy in patients at risk to develop disseminated disease, and for the documentation of the efficacy of any treatment modality. Data on pharmacokinetics of ribavirin and cidofovir are required, especially in children after SCT, to document that a lack of efficacy is not due to the relatively low level of active components. In vitro susceptibility testing of clinical isolates obtained prior to initiation and after finishing of treatment will be instrumental in evaluating the in vivo effect of treatment.

Based on current knowledge, the efficacy of antiviral medication in immunocompromised patients is disputable and boosting of immunity by tapering of immunosuppression or infusion of cells from the donor seems an essential element in the treatment of patients at risk of HAdV viraemia. To lower the risk of alloreactivity, methods for allodepletion of donor T cells should be improved. Enrichment for HAdV-specific T cells using in vitro culture systems and conserved HAdV-derived peptides appears a promising and feasible alternative. Various research groups are currently investigating the role of CD4+ and CD8+ T cells in the cellular immune response to HAdV.23, 25, 26, 27 Together with a more detailed analysis of the origin of the infectious HAdV in the setting of allogeneic SCT and of the HAdV type(s) infecting a patient, this research will certainly enhance our knowledge of the interaction between HAdV and the immune system. This will be of benefit not only for recipients of stem cell grafts but also for recipients of solid organ transplants and other immunocompromised patients in which HAdV infection may have serious complications. In addition, knowledge about the interaction between the immune system and HAdV may have an impact on the results of gene therapy strategies using HAdV-based vectors.

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Correspondence to M J D van Tol.

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van Tol, M., Claas, E., Heemskerk, B. et al. Adenovirus infection in children after allogeneic stem cell transplantation: diagnosis, treatment and immunity. Bone Marrow Transplant 35, S73–S76 (2005). https://doi.org/10.1038/sj.bmt.1704852

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Keywords

  • adenovirus infection
  • allogeneic SCT
  • children
  • diagnosis
  • treatment
  • immunity

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