Human Cytomegalovirus Latency and Reactivation in Allogeneic Hematopoietic Stem Cell Transplant Recipients

1Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by a majority of the global population. The seroprevalence of HCMV increases with age and has an estimated global mean of 83%. While primary infection is usually asymptomatic in immunocompetent individuals, the virus establishes a lifelong latent infection that is never eliminated by the host immune system. Intermittent subclinical viral reactivation events are thought to be controlled by effective immune surveillance and may drive the high frequencies of HCMV-specific T-cells found in the peripheral blood of healthy seropositive individuals. By contrast, reactivation from latency is responsible for significant morbidity and mortality in immunocompromised and immunosuppressed populations,including solid-organ transplant and allogeneic hematopoietic stem cell transplant (HSCT) recipients, HIV/AIDS patients and the developing fetus.

2HCMV reactivation is the major viral infectious complication after allogeneic HSCT and is associated with an increased risk of non-relapse mortality, which is not principally attributable to direct HCMV disease. Uncontrolled HCMV replication following reactivation can lead to life-threatening end-organ disease, commonly manifesting as pneumonia and gastrointestinal disease, and less frequently as retinitis, hepatitis and encephalitis. Reactivation may also indirectly contribute to detrimental patient outcomes through antiviral drug toxicities and complex impacts on post-transplant immune reconstitution, including links with graft-versus-host disease (GvHD) and microbial superinfections. There is no licensed HCMV vaccine and current antiviral agents are limited by their toxic side-effects and the risk of antiviral drug resistance.

3The cellular sites and mechanisms associated with HCMV latency and reactivation are incompletely understood, in part due to the high human-specificity of HCMV which precludes the use of animal models to directly study HCMV infection. Clinical evidence indicates that the risk of reactivation post-HSCT is strongly connected to the pre-transplant HCMV serostatus of the donor and recipient and the pace of post-transplant HCMV-specific T-cell recovery. However, the lack of well-validated biomarkers for reactivation makes it challenging to predict the incidence and outcome of infection in individual patients, and greater knowledge of the influence of HCMV on post-transplant immune reconstitution is required.

4Allogeneic HSCT is the only curative option for many hematological malignancies and diseases. GvHD, opportunistic infections and relapse are the leading causes of mortality in the first 2 years post-transplant. The reactivation of latent double-stranded DNA viruses is common in the early post-engraftment period and HCMV reactivation is the most frequent opportunistic viral infection after HSCT. Virological surveillance for HCMV replication is routinely performed in the first 100 days post-transplant through quantitative PCR monitoring for HCMV genomes in blood plasma, with HCMV DNAemia being an independent risk factor for disease and non-relapse mortality.

5Reactivation develops in over 60% of HCMV seropositive recipients (R+), and in approximately 10% of seronegative recipients (R-) transplanted from seropositive donors (D+). In R+/D+ patients, reactivation may derive from endogenous latent HCMV in the seropositive recipient and/or from latently infected cells transferred within the seropositive donor stem cell allograft. Recipient seropositivity alone is an adverse prognostic factor for overall survival. Seropositive recipients who receive grafts from HCMV-naïve donors (R+/D-) experience the highest incidence of HCMV reactivation and disease, a likely consequence of delayed HCMV-specific immune recovery owing to the lack of pre-existing HCMV-specific memory lymphocytes in the graft. As the recipient’s existing cellular immune system is eradicated by transplant conditioning regimens, R+/D- patients rely on de novo T-cell reconstitution via the thymus from donated pluripotent hematopoietic stem cells to generate a primary T-cell response to HCMV reactivation. By contrast, HCMV-specific memory T-cells contained in D+ grafts can undergo more rapid antigen-driven expansion within the recipient in the early post-transplant period and contribute to early control of reactivation. For a seropositive recipient, the choice of a seropositive over seronegative donor offers a survival advantage in the setting of myeloablative unrelated donor transplantation, but the effect on survival with matched sibling donors is less strong. Nonetheless, due to the high incidence of HCMV-related complications in R+/D- patients, where possible, attempts are made to match serostatus in donors and recipients.

6Advances in transplant and antiviral treatment practices over the last 25 years have reduced the incidence of HCMV disease to ∼10% in the first year post-transplant. However, the use of prophylactic or pre-emptive antiviral therapy delays HCMV-specific T-cell recovery and has led to increasing rates of late HCMV reactivation and disease. Additionally, HCMV pneumonia remains associated with high (up to 70%) mortality. Gastrointestinal HCMV disease often develops without detection of HCMV DNAemia and can be difficult to distinguish from gastrointestinal GvHD, often occurring in the same patients.

7The transition from viral latency to reactivation underpins the pathogenesis of HCMV in HSCT. HCMV latency is characterized by maintenance of the viral genome as an intranuclear episome without replication, but with the potential to reactivate to a productive infection. A wide range of cell types support productive infection, but latency appears to be restricted to primitive bone-marrow-resident CD34+ cells and CD33+ myeloid progenitor cells, which retain the latent viral genome as they differentiate into peripheral blood CD14+ monocytes and myeloid dendritic cells (mDCs). A recent study found that CD14+ monocytes expressing the surface marker B7-H4 were a predominant site of latency in peripheral blood of healthy donors. It may be that HCMV preferentially infects early myeloid progenitors or promotes the differentiation of infected pluripotent CD34+ cells to myeloid-lineage subsets that support latency.

8Latently infected cells contain HCMV DNA but do not support infectious virus production. The terminal differentiation to mature mDCs and macrophages is accompanied by chromatin remodeling of the HCMV major immediate-early promoter, which facilitates reactivation of the lytic gene cascade and the production of infectious virus. Allogeneic stimulation and pro-inflammatory cytokines such as IFN-γ, TNF, and IL-6 are implicated in driving myeloid cell maturation and reactivation.

9Latently infected cells are present at very low frequencies (0.004–0.01% of mononuclear cells) in GCSF-mobilized peripheral blood or bone-marrow from healthy seropositive donors, but underlie the capacity for iatrogenic transmission of latent HCMV through D+ HSCT allografts. Additionally, the high risk of reactivation in R+/D- patients suggests that pre-transplant conditioning regimens incompletely eradicate latent HCMV reservoirs in the recipient. It also remains possible there are additional sites of HCMV latency, with conflicting evidence regarding possible latency in aortic endothelial cells. Whether HCMV establishes a low-level productive infection in bone-marrow stromal cells or in a self-renewing CD34+ cell subset also remains unclear, yet HCMV DNA has been detected in diverse tissue sites and recent RNA-seq uncovered HCMV transcripts at multiple locations, including the ovaries, blood, adipose tissue, and lung. The specific cell types harboring HCMV in these studies and whether they represent productive, abortive, or latent infection is unknown. The widespread prevalence of HCMV within asymptomatic individuals nonetheless highlights the importance of host immune control in preventing unchecked HCMV replication leading to life-threatening disease.

10In addition to recipient and donor HCMV serostatus, independent risk factors for reactivation include increasing recipient age, use of unrelated or HLA-mismatched donors, T-cell depletion, GvHD, and high-dose corticosteroids for GvHD. T-cell depletion and prolonged steroid therapy mitigate GvHD but delay antiviral T-cell reconstitution. A high incidence of HCMV reactivation is also observed after T-cell replete haploidentical HSCT with post-transplant cyclophosphamide.

11The reconstitution kinetics of HCMV-specific T-cells post-HSCT have a close relationship with the risk and prognosis of reactivation. HCMV-specific CD4+ and CD8+ T-cells expand with reactivation and are likely both required for control and/or protection. Quantitative thresholds of CD8+ and CD4+ HCMV-specific T-cells associated with protection from, or control of, reactivation or disease post-HSCT have been defined using HLA tetramers or ex vivo viral stimulation assays. Threshold numbers have not been well-validated for routine clinical use and are less informative of protection from reactivation and disease in patients under steroid therapy or with prior GvHD.

12Control of reactivation may depend more heavily on the functional recovery of HCMV-specific T-cell immunity. Polyfunctional HCMV-specific T-cell responses post-HSCT are associated with lower viral loads, protection from subsequent episodes of reactivation and lower antiviral therapy requirements. Delayed or undetectable HCMV-specific cytotoxic T-cell responses are prominent risk factors for HCMV disease. recently described a composite biomarker comprising a protective (IL-2+IFN-γ+TNF-α+MIP-1β+) and non-protective (IL-2-IFN-γ+TNF-α-MIP-1β+) CD8+ T-cell cytokine signature in response to in vitro HCMV pp65 peptide stimulation that independently predicted the risk of clinically significant reactivation. Assessment of HCMV-specific immunity through measurement of whole blood ex vivo IFN-γ secretion responses to HCMV peptides has also emerged as a promising prognostic approach for HCMV reactivation post-HSCT. However, it is argued that the selective recovery of HCMV-specific T-cell immunity ahead of global T-cell reconstitution carries a higher risk of subsequent reactivation.

13The detection of reactivation prior to 100 days post-transplant, plasma viral load, leukopenia, lymphopenia, and GvHD represent additional risk factors for HCMV disease. Donor grafts with ≥5 activating killer-cell immunoglobulin-like receptor (KIR) genes or with both KIR2DS2 and KIR2DS4 predict a lower risk of reactivation, and the use of donors with multiple or additional activating KIRs is associated with a lower incidence of reactivation.

14The immune system crucially regulates the risk of HCMV reactivation and disease, but HCMV itself also has a profound influence in shaping immune profiles in healthy seropositive individuals and HSCT patients, although the functional implications of this immune modulation are not yet clear. In some HSCT recipients, reactivation might be an epiphenomenon of poor immune reconstitution, but it is also possible that the immunomodulatory effects of HCMV infection may contribute to poor post-transplant outcomes. Indeed, while HCMV DNAemia-guided pre-emptive antiviral therapy has reduced the incidence of HCMV disease post-HSCT, the survival disadvantage associated with HCMV infection has not been eliminated. HCMV encodes a range of immunomodulatory gene products that are expressed during both productive infection and latency, including a homolog of the immunosuppressive cytokine IL-10. HCMV infection in HSCT recipients increases the risk of bacterial and fungal superinfections and GvHD, which might be connected to complex impacts of HCMV reactivation and/or its treatment on post-transplant immune recovery.

15Reactivation stimulates the recovery of HCMV-specific T-cells after HSCT (see section “Risk Factors for HCMV Reactivation After HSCT”), but is also accompanied by broader changes in the T-cell compartment. Patients with reactivation display accelerated CD8+ T-cell reconstitution in the first year post-transplant, which is likely to be driven by clonal expansions of HCMV-specific effector-memory αβ CD8+ T-cells, leading to an inverted CD4:CD8 ratio. Deep sequencing of the TCR-β repertoire at 1 year post-HSCT uncovered a contraction in effector-memory CD8+ TCR diversity and holes in the underlying effector-memory CD8+ T-cell repertoire in patients who had experienced reactivation. The selective expansion of HCMV-reactive Vδ2neg γδ T-cells, and clonal (Vγ9neg and Vδ2neg) γδ T-cell proliferations suggestive of adaptive responses, are also observed following reactivation. Reactivation also triggers the expansion of large granular lymphocytes. found the reconstitution of naïve and central memory T-cells up to 2 years post-transplant was not compromised by reactivation, but observed lower numbers of naïve T-cells in the first year post-HSCT in patients with reactivation. Many of the immunological features associated with HCMV reactivation after HSCT, including oligoclonal expansions of terminally differentiated HCMV-specific T-cells, are also found with aging in seropositive individuals, although recently it was reported that these HCMV-induced clonal T-cell expansions may not compromise CD8+ T-cell repertoire diversity in the elderly. The prominent influence of HCMV seropositivity and reactivation in shaping global immune reconstitution signatures after HSCT is apparent, but the impact of reactivation on immune recovery beyond 2 years post-transplant is not well-characterized.

16HCMV reactivation drives a rapid expansion of IFN-γ-producing NKG2C+ NK cells, which likely contribute to early control of reactivation. Expanded proportions of mature (CD56dimCD57+NKG2A-CD158b+) NKG2C+ NK cells persist after viral clearance and memory-like expansions of NKG2C+CD57+ NK cells are also observed in R+/D+ patients with subclinical HCMV infection. Specific recognition by NKG2C+ NK cells of HCMV UL40 peptides presented in the context of HLA-E was recently identified to be the mechanism that drives the expansion and differentiation of NKG2C+ NK cells during HCMV infection.

17An association between early HCMV reactivation and reduced myeloid leukemia relapse has been reported. This putative protective effect might be mediated through the anti-leukemic activities of CD56dimCD57+NKG2C+ NK cell and Vδ2neg γδ T-cell subsets which expand with reactivation, or via enhancement of donor alloimmune responses in the presence of infection and HCMV-specific CD8+ T-cells. However, the role of reactivation in protection from malignancy relapse post-HSCT is controversial, as others have not found evidence of this association in patients with acute myeloid leukemia, chronic myeloid leukemia, acute lymphoid leukemia, lymphoma, or myelodysplastic syndrome. Further studies are thus required to better define the patient subgroups and immunological features associated with possible relapse protection in HSCT patients with HCMV reactivation.

18Graft-versus-host disease and its steroid therapy increase the risk of reactivation after HSCT. The alloimmune responses mediating GvHD impair thymopoiesis and delay HCMV-specific T-cell reconstitution, and high-dose steroids impair the recovery of HCMV-specific T-cells in a dose-dependent manner. Further, it has been speculated that the proinflammatory immune environment associated with GvHD may promote reactivation, as has been demonstrated following allogeneic stimulation of latently infected cells ex vivo. Conversely, patients with reactivation more frequently develop GvHD and the risk of extensive chronic GvHD was reduced with the use of HCMV DNAemia-guided pre-emptive antiviral therapy. An increased risk of acute GvHD was observed during episodes of active HCMV replication after HSCT, supporting a bidirectional relationship between reactivation and GvHD. Further research is required to delineate the mechanisms underlying this phenomenon, but an inflammatory response to reactivation or the potential cross-reactivity of HCMV-specific T-cells with human alloantigens might play a role.

19Standard antiviral drugs for reactivation after HSCT are ganciclovir, valganciclovir and foscarnet. Prophylactic use is reserved for high-risk patients due to their significant toxic side effects. Ganciclovir causes neutropenia which increases the risk of bacterial and fungal superinfections, while foscarnet and cidofovir (used as a second- or third- line therapy) are nephrotoxic. Additional concerns relate to the development of antiviral drug resistance and breakthrough reactivation or disease. HCMV drug resistance has been reported in up to 14.5% of HSCT recipients receiving pre-emptive therapy. Prolonged antiviral exposure, suboptimal prophylactic dosing, corticosteroid therapy and delayed immune reconstitution foster the selection of drug resistant HCMV mutants. Mutations in the HCMV UL97 (viral protein kinase) gene confer resistance to (val)ganciclovir and maribavir, while HCMV UL54 (viral DNA polymerase) gene mutations are associated with resistance to foscarnet, cidofovir and (val)ganciclovir. Infection with multiple HCMV genotypes is associated with reduced efficacy of antiviral treatment. Importantly, current drugs do not target HCMV during latency, as these drugs target the viral replication machinery, and latency is typified by maintenance of the viral genome without replication. This highlights the potential for clinically relevant recurrence of reactivation following therapy cessation.

20Prophylactic administration of Letermovir, a new anti-HCMV agent which inhibits the viral terminase complex, recently demonstrated the capacity to reduce the risk of HCMV disease and all-cause mortality at 24 weeks post-transplant, in a Phase 3 trial. There are, however, reports emerging of breakthrough viraemia and disease associated with HCMV UL56 terminase mutations conferring Letermovir resistance in HSCT recipients. Letermovir has now been licensed for HCMV prophylaxis after HSCT and its efficacy in treating refractory or resistant HCMV infection and disease will be evaluated in an upcoming Phase 2 trial (NCT03728426). Given its unique mechanism of action, combination therapy of Letermovir with other currently licensed antivirals may represent a means to more effectively control HCMV and limit the emergence of antiviral drug resistance in HSCT patients, although this area remains to be explored.

21Pre-emptive treatment based on viral DNAemia surveillance minimizes toxic drug exposure and reduces rates of HCMV disease and mortality, but there is no consensus on the appropriate plasma viral load threshold for initiating such therapy. Inter-laboratory assays for HCMV DNA quantitation vary and the WHO reference standard for HCMV DNA lacks commutability in many assays. Lower viral load thresholds may be required in settings of corticosteroid treatment and T-cell depletion. Monitoring both HCMV-specific T-cell immunity and viral load has recently been successfully applied to guide the withholding or early discontinuation of antiviral treatment. Further to characterizing immune reconstitution profiles in patients who spontaneously resolve reactivation without antiviral treatment, more detailed investigation of the immune environment prior to the detection of HCMV DNAemia should be a focus of future studies to optimize the identification of high-risk patients and timing of pre-emptive therapy.

22Pooled HCMV-specific or polyclonal intravenous immunoglobulin is not effective at preventing reactivation or reducing mortality when used in the treatment of HCMV pneumonia post-HSCT, although strain-specific antibody therapy was recently shown to potently inhibit murine cytomegalovirus (MCMV) reactivation after bone-marrow transplantation in a preclinical murine model. Adoptive HCMV-specific T-cell therapies to prevent and treat reactivation after HSCT have been developed since the early 1990s. Third-party- or stem cell donor-derived HCMV-specific T-cells expanded ex vivo or isolated directly with HCMV-specific tetramers and infused in the post-transplant period can accelerate HCMV-specific immune recovery and contribute to long-term control of reactivation and protection from HCMV disease. The post-transplant infusion of donor-derived HCMV-specific cytotoxic T-cells was shown to reduce the requirement for antiviral drug therapy in a Phase 2 trial. Further studies are needed to determine the optimal timing of adoptive cell infusion and understand its impact on post-transplant immune reconstitution.

23HCMV is a highly prevalent, opportunistic pathogen that continues to cause substantial morbidity and mortality after HSCT. Improved knowledge of the cellular sites of HCMV latency and the conditions which enable its reactivation to clinically significant infection will be needed to better predict, prevent and control reactivation post-HSCT. Future strategies might involve the selective depletion of latently infected cells from the graft, plasma metabolomics profiling to predict the emergence of reactivation, the vaccination of transplant recipients and donors to enhance HCMV-specific immune reconstitution, or the engineering of corticosteroid-resistant HCMV-specific T-cells to improve adoptive cell therapies. The marked impact of HCMV on post-transplant immune reconstitution warrants continued research to understand its relationship with patient outcomes. New therapeutic approaches for reactivation are actively being pursued and it is hoped these will lessen the clinical impact of reactivation after HSCT in the near future.

24LS and BW generated the initial draft of the manuscript. All authors contributed to the subsequent writing and review of the manuscript.

25The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.