Epstein-Barr Virus Reactivation Research Articles

Letermovir prophylaxis reduced cytomegalovirus reactivation and resistance post umbilical cord blood transplantation.

To explore the impact of letermovir (LET) prophylaxis on cytomegalovirus (CMV) reactivation and resistance in both adult and paediatric umbilical cord blood transplantation (UCBT) patients, we retrospectively compared 43 UCBT patients who received LET as CMV prophylaxis with a historical cohort of 207 UCBT patients without LET usage. LET was administered from Day +1 to Day +100. The 180-day cumulative incidence of CMV reactivation (47.3% vs. 74.4%, p < 0.001) and the proportion of refractory CMV reactivation (15.0% vs. 42.9%, p = 0.016) were significantly lower than those in the control group. However, more frequent late CMV infection (31.0% vs. 4.3%, p = 0.002) and the 180-day cumulative incidence of Epstein-Barr virus (EBV) reactivation (9.3% vs. 3.4%, p = 0.087) were observed in UCBT patients with LET prophylaxis. Meanwhile, older age (>15 years old) and the occurrence of pre-engraftment syndrome were identified as the significant risk factors for CMV reactivation, and in patients at high risk, the incidence of CMV reactivation in the LET group was lower than that in the control group (46.7% vs. 86.5%, p < 0.001), while this decline was less pronounced among patients at low risk (47.8% vs. 62.1%, p = 0.120).

Abstract 5786: Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC

Abstract Introduction: The prevailing histologic subtype of nasopharyngeal carcinoma (NPC) in the endemic regions is non-keratinizing carcinoma which shows almost consistent association with latent infection by Epstein-Barr virus (EBV). Previously our group developed a series of Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes, which can disrupt the EBV latency maintained by EBNA1, and such lytic induction could alert the immune cells. Methods: Natural killer (NK) cells of the innate immunity play a significant role in controlling EBV infection. In this study, we investigated whether our EBNA1 probes (ZRL5P4 &amp; L2P4) could activate NK cells to eliminate NPC. Results: Studies of clinical specimens indicated that tumor infiltration of the cytotoxic NK cell subset is a favorable prognostic marker for NPC patients. Furthermore, the cytotoxicity of circulating NK cells derived from NPC patients is equally well the cells from healthy individuals. If those patients’ NK cells can be attracted to infiltrate into the tumors, the tumor burden should be relieved. Indeed, the NPC xenograft with the ZRL5P4 treatment showed heavy mouse NK cell infiltration and tumor size reduction. The essential role of those mouse NK cells was demonstrated. Next, we showed that ZRL5P4 could induce the production of a soluble factor by NPC cells to stimulate the NK cell cytotoxicity. Such NK cell activity was verified in a metastasis NPC model. Mechanistically, the EBNA1 probe treatment could induce the expression of EBV early lytic gene Rta and EBNA1 itself. Both Rta and EBNA1 are known transcription factors to drive the expression of LMP1, the NF-κB signaling was then activated, which turned on the production of the well-known NK cell activation cytokine IFN-β. Conclusion: Taken together, our results demonstrated that our EBNA1 probes can mobilize NK cells to the tumor sites and activate their anti-tumor activity via the EBV reactivation and stimulation of IFN-β production. Citation Format: Luo Chen, Xiao Han Liu, Hei Tung Yuen, William C. Cho, Ka-Leung Wong, Hong Lok Lung. Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5786.

Adoptive transfer of donor B lymphocytes: a phase 1/2a study for patients after allogeneic stem cell transplantation

AbstractImmune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is slow and patients carry a high and prolonged risk of opportunistic infections. We hypothesized that the adoptive transfer of donor B cells can foster after HSCT immuno-reconstitution. Here, we report, to our knowledge, the results of a first-in-human phase 1/2a study aimed to evaluate the feasibility and safety of adoptively transferred donor B cells and to test their activity upon recall vaccination. Good manufactoring practice (GMP) B-cell products were generated from donor apheresis products using 2-step magnetic cell separation. Fifteen patients who had undergone allo-HSCT were enrolled and treated after taper of immunosuppression (median, day +148; range, 130-160). Patients received 4 different doses of B cells (0.5 × 106 to 4.0 × 106 B cells per kg body weight). To test the activity of infused donor memory B cells in vivo, patients were vaccinated with a pentavalent vaccine 7 days after B-cell transfer. We observed the mobilization of plasmablasts and an increase in serum titers against vaccine antigens, with a stronger response in patients receiving higher B-cell numbers. Analysis of immunoglobulin VH-sequences by next-generation sequencing revealed that plasmablasts responding to vaccination originated from memory B-cell clones from the donor. Donor B-cell transfer was safe, as no Epstein-Barr virus (EBV) reactivation was observed, and only low-grade graft-versus-host disease (GVHD) occurred in 4 out of 15 patients. This pilot trial may pave the way for further studies exploring the adoptive transfer of memory B cells to reduce the frequency of infections after allo-HSCT. This trial was registered at ClinicalTrial.gov as #NCT02007811.

Recombinant Human Thrombopoietin Promotes Platelet Engraftment in Severe Aplastic Anemia Patients Following Treatment With Haploid Hematopoietic Stem Cell Transplantation using Modified Post-Transplantation Cyclophosphamide

BackgroundRecombinant human TPO (rhTPO) promotes platelet engraftment in patients after allogeneic HSCT (allo-HSCT). However, the effects of rhTPO on platelet recovery after Haplo-HSCT in patients with severe aplastic anemia (SAA) have not been intensively studied. ObjectiveWe aimed to evaluate the efficacy of rhTPO on platelet engraftment in patients with SAA who were treated with Haplo-HSCT using post-transplantation cyclophosphamide (PTCy). Study DesignSAA patients who received Haplo-HSCT plus PTCy regimen were divided into the rhTPO group (with subcutaneous injection of rhTPO, n = 28) and Control group (no rhTPO administration, n = 27). The engraftment of platelet/neutrophil, platelet infusion amount, and transplant-related complications between the 2 groups were compared. ResultsAll 55 patients showed successful hematopoietic reconstitution. The median time of platelet engraftment was 11 (9 to 29) days in the rhTPO group and 14 (9 to 28) days in the Control group (P = .003). The rhTPO group had a significantly reduced amount of infused platelets compared to the Control group (2 (1 to 11.5) versus 3 (1 to 14) therapeutic doses; P = .004). There was no significant difference between the 2 groups regarding median time of neutrophil engraftment, incidence of acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD), incidence of cytomegalovirus or Epstein–Barr virus reactivation, 3-yr overall survival rate, and failure-free-survival rate. No obvious adverse reactions were observed in the rhTPO group. ConclusionrhTPO promoted platelet engraftment, reduced the amount of transfused platelets, and demonstrated good safety profiles without evidence of adverse reactions in patients with SAA who received Haplo-HSCT using PTCy regimen.

[Clinical study of the efficacies of ruxolitinib plus low-dose PTCY for acute GVHD prevention after haploidentical transplantation in malignant hematological diseases].

Objective: To investigate and verify a novel acute graft versus host disease (aGVHD) prevention protocol in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) . Methods: Patients who underwent haplo-HSCT in our center between January 2022 and December 2022 were included. All patients received reduced doses of cyclophosphamide, Rabbit anti-human tymoglobulin, ruxolitinib, methotrexate, cyclosporine, and MMF to prevent aGVHD. The transplantation outcomes, complications, and survival rate of all patients were investigated. Results: A total of 52 patients with haplo-HSCT were enrolled, 29 (55.8%) male and 23 (44.2%) female, with a median age of 28 (5-59) years. There were 25 cases of acute myeloid leukemia, 17 cases of acute lymphocyte leukemia, 6 cases of myelodysplastic syndrome, 2 cases of chronic myeloid leukemia and 2 cases of myeloproliferative neoplasms. 98.1% of patients had successful engraftment. The incidence of Ⅱ-Ⅳ aGVHD and Ⅲ-Ⅳ aGVHD was 19.2% (95% CI 8.2% -30.3% ) and 7.7% (95% CI 0.2% -15.2% ), respectively. No patients experienced severe gastrointestinal mucositis. The Epstein-Barr virus and CMV reactivation rates were 40.4% and 21.3%, respectively. 9.6% of patients relapsed during followup, with 1-year overall survival, progression-free survival, and non-relapse mortality rates of 86.5% (95% CI 76.9% -96.1% ), 78.8% (95% CI 67.4% -90.3% ) and 11.5% (95% CI 2.6% -20.5% ), respectively. Conclusion: Ruxolitinib combined with a low dose of PTCY is a safe and effective first-line aGVHD prevention strategy.

BGLF4 kinase regulates the formation of the EBV cytoplasmic assembly compartment and the recruitment of cellular IQGAP1 for virion release.

After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.

Early T-cell reconstitution predicts risk of EBV reactivation after allogeneic hematopoietic stem cell transplantation

The quality of immune reconstitution (IR) is crucial for the outcome of patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is closely connected with infection, relapse and graft-versus-host disease (GvHD) which are the most important causes for transplantation failure. However, the IR pattern in the early stage after allo-HSCT, particularly haploidentical (HID) HSCT, remains unclear. In this retrospective study, we examined the T cell reconstitution of patients within the initial 30 days (n = 173) and 100 days (n = 122) after allo-HSCT with myeloablative condition (MAC), of which > 70% were HID HSCT, to assess the influence of IR on the transplant outcomes. By comparing 78 patients with good IR (GIR) to 44 patients with poor IR (PIR), we observed that GIR was associated with lower risk for Epstein–Barr virus (EBV) reactivation and cytomegalovirus (CMV) reactivation, but had no significant impacts on the survival outcomes (i.e., overall survival, event-free survival) and cumulative incidences of GvHD. Importantly, we found lymphocyte reconstitution pattern at day 30 after allo-HSCT would be a surrogate for IR evaluated at day 100. In the Cox proportional hazard model, early reconstitution of CD4+, CD4+CD25+, CD4+CD45RO+, CD4+CD25+CD27low, and CD8+ T cells at day 30 was reversely correlated with risk of EBV reactivation. Finally, we constructed a predictive model for EBV reactivation with CD8+ and CD4+CD45RO+ T cell proportions of the training cohort (n = 102), which was validated with a validation cohort (n = 37). In summary, our study found that the quality of IR at day 30 had a predictive value for the risk of EBV reactivation, and might provide guidance for close monitoring for EBV reactivation.

EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation.

Epstein-Barr virus (EBV) is associated with several types of human cancer including nasopharyngeal carcinoma (NPC). The activation of EBV to the lytic cycle has been observed in advanced NPC and is believed to contribute to late-stage NPC development. However, how EBV lytic cycle promotes NPC progression remains elusive. Analysis of clinical NPC samples indicated that EBV reactivation and immunosuppression were found in advanced NPC samples, as well as abnormal angiogenesis and invasiveness. To investigate the role of the EBV lytic cycle in tumor development, we established a system that consists of two NPC cell lines, respectively, in EBV abortive lytic cycle and latency. In a comparative analysis using this system, we found that the NPC cell line in EBV abortive lytic cycle exhibited the superior chemotactic capacity to recruit monocytes and polarized their differentiation toward tumor-associated macrophage (TAM)-like phenotype and away from DCs, compared to EBV-negative or EBV-latency NPC cells. EBV-encoded transcription activator ZTA is responsible for regulating monocyte chemotaxis and TAM phenotype by up-regulating the expression of GM-CSF, IL-8, and GRO-α. As a result, TAM induced by EBV abortive lytic cycle promotes NPC angiogenesis, invasion, and migration. Overall, this study elucidated the role of the EBV lytic life cycle in the late development of NPC and revealed a mechanism underlying the ZTA-mediated establishment of the tumor microenvironment (TME) that promotes NPC late-stage progression.

Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

Understanding the Mode of Action of a Micro-Immunotherapy Formulation: Pre-Clinical Evidence from the Study of 2LEBV® Active Ingredients.

Epstein-Barr virus (EBV) is often kept silent and asymptomatic; however, its reactivation induces a chronic and/or recurrent infection that is associated with numerous diseases, including cancer and inflammation-related disorders. As no specific treatment is currently available, the immune factors-based micro-immunotherapy (MI) medicine 2LEBV® could be considered a valuable therapeutic option to sustain the immune system in EBV reactivation. The present work aimed to investigate, for the first time, the effect of 2LEBV® in several in vitro models of uninfected immune-related cells. 2LEBV® displayed phagocytosis-enhancing capabilities in granulocytes. In human peripheral blood mononuclear cells (PBMCs), it increased the intra- and extra-cellular expression of interleukin (IL)-2. Moreover, it modulated the secretion of other cytokines, increasing IL-4, IL-6, and tumor necrosis factor-α levels or lowering other cytokines levels such as IL-9. Finally, 2LEBV® reduced the expression of human leukocyte antigen (HLA)-II in endothelial cells and macrophages. Although these data are still preliminary and the chosen models do not consider the underlying EBV-reactivation mechanisms, they still provide a better understanding of the mechanisms of action of 2LEBV®, both at functional and molecular levels. Furthermore, they open perspectives regarding the potential targets of 2LEBV® in its employment as a therapeutic intervention for EBV-associated diseases.

Rare Co-occurrence of Spinal Cord Hemorrhage from Radiation-induced Cavernous Hemangioma and Classical Hodgkin Lymphoma Post-transplant Lymphoproliferative Disorder.

Post-transplant lymphoproliferative disorders (PTLDs) are lymphoproliferative diseases that occur after solid organ transplantation or hematopoietic stem cell transplantation (HSCT). The development of PTLD is often associated with reactivation of Epstein-Barr virus (EBV). A 26-year-old woman with a history of HSCT and total-body irradiation developed spinal cord hemorrhage from a radiation-induced cavernous hemangioma (RICH) shortly after the development of classical Hodgkin lymphoma PTLD with EBV reactivation. Although little is known about the factors leading to hemorrhagic events from spinal cord RICH, we suspect that EBV reactivation may have been a factor contributing to the hemorrhage in the present case.

Clinical characteristics and outcomes of Epstein-Barr virus viral load after allogeneic hematopoietic stem cell transplantation

Epstein-Barr virus (EBV) reactivation can occur following allogenic hematopoietic stem cell transplantation (allo-HSCT). However, the clinical characteristics and outcomes of EBV-viral load are not well known. Thus, we retrospectively analyzed the clinical features and prognostic impact of the EBV viral load in 121 allo-HSCT recipients from our hospital. EBV DNA quantification was performed in whole blood after transplantation. Patients were grouped based on whether EBV DNA quantification reached > 1000 copies/mL during follow-up (N = 50) or not (N = 71). Patients with EBV > 1000 EBV copies/mL were relatively more common in the groups with graft versus host disease (GVHD) prophylaxis including ATG, haploidentical donor type, peripheral blood as a donor source, and acute GVHD II–IV. The 20-month OS and DFS were not significantly different between patients with < 1000 EBV copies/mL and patients with > 1000 EBV copies/mL (20-month OS, 56.0% vs. 60.6%; p = 0.503, 20-month DFS, 50.0% vs. 57.7%; p = 0.179). Immunosuppressant (ISS) dose reduction was achieved after the maximum increase in EBV in 41/50 (82%) patients. Additionally, 30/50 (60%) patients achieved a 50% dose reduction or no restarting of ISS within 3 months of the maximum EBV increase. Among cases wherein EBV DNA quantification reached > 1000 copies/mL, those that achieved rapid dose reduction of ISS tended to have longer overall survival (“not reached” vs 5.4 months, p < 0.001) and disease-free survival (88.4 months vs 5.3 months, p < 0.001) than those in patients who did not. Our data highlight the importance of rapid ISS reduction in post-transplant EBV reactivation.

Host factor KAP1 coordinates temporal control between transcription and replication

Temporal control of transcription and replication is necessary for efficient Epstein–Barr virus reactivation. Xu et al. identified the KAP1/EA-D/ATM axis as a critical regulator of these processes. This discovery illuminates the collaboration between host and viral factors as an essential interaction for viral reactivation.

Donor aKIR genes influence the risk of EBV and CMV reactivation after anti-thymocyte globulin-based haploidentical hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) offers the highest curative potential for patients with hematological malignancies. Complications including infection, graft-versus-host disease (GVHD), and relapse reflect delayed or dysregulated immune reconstitution. After transplantation, NK cells rapidly reconstitute and are crucial for immune surveillance and immune tolerance. NK cell function is tightly regulated by killer immunoglobin-like receptors (KIRs). Previous studies have revealed that donor KIRs, especially some activated KIRs (aKIRs) are closely related to transplant outcomes. Here, we performed a retrospective study, including 323 patients who received haploidentical (haplo) HSCT in our center. In univariate analysis, donor KIR2DS1, KIR2DS3 and KIR3DS1 gene protected patients with lymphoid disease from Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reactivation, while donor KIR2DS1, KIR2DS5 and KIR3DS1 gene conferred a higher risk of CMV reactivation for patients with myeloid disease. Multivariate analysis confirmed that donor telomeric (Tel) B/x and KIR2DS3 gene best protected patients with lymphoid disease from EBV (p = 0.017) and CMV reactivation (p = 0.004). In myeloid disease, grafts lacking Tel B/x and KIR2DS5 gene correlated with the lowest risk of CMV reactivation (p = 0.018). Besides, donor aKIR genes did not influence the rates of GVHD, relapse, non-relapse mortality (NRM) and overall survival (OS) in this study. The reactivation of EBV and CMV was associated with poor prognosis of haplo-HSCT. In conclusion, we found that donor aKIR genes might have a synergistic effect on CMV and EBV reactivation after haplo-HSCT. Whether the influence of donor aKIR genes varies with disease types remained to be studied.

EBV Reactivation in Transplant Recipients following SARS-CoV-2 Infection: A Retrospective Study.

Reactivation and primary infection with a high Epstein Barr Virus (EBV) DNA level in kidney transplant patients could cause severe complications, including the development of Post-Transplantation Lymphoproliferative Disease (PTLD). While in the general population the reactivation of EBV after SARS-CoV-2 infection has been reported, very few data are available in transplant recipients. Our retrospective study aimed to evaluate a possible EBV reactivation in kidney transplant patients following SARS-CoV-2 infection and a possible impairment of the immune system. In addition, the effects of changes in immunosuppressive therapy on EBV DNA reactivation and vaccination were also evaluated. A total of 166 kidney transplant patients followed at the Kidney-Pancreas Transplant Ambulatory Nephrology Unit at Padova University Hospital were retrospectively considered for an observation period of 6 months from January 2020 to April 2023. EBV DNA level was measured by Rt-PCR and evaluated 6 months before and after SARS-CoV-2 infection. Patients' serological states were established via quantification of anti-VCA and anti-EBNA (chemiluminescence). Patients' immune systems were characterized by CD4+/CD8+ lymphocyte ratio (flow cytometry). EBV DNA was reactivated in 50% of the 166 patients with COVID-19 who completed the study. Older patients with more severe forms of COVID-19 had higher EBV reactivation (p < 0.05). EBV reactivation significantly increased in patients with severe SARS-CoV-2 infection requiring hospitalization compared to patients managed at home (p < 0.001). CD4+/CD8+ lymphocyte ratio was reduced in patients with a younger age of transplant (p < 0.01) and on a higher dose of steroids (p < 0.01). The results of our study confirm the role of immunodepression, especially in recent transplant patients and those on high steroids, in EBV reactivation. These results combined with the few available in the literature might contribute to providing an optimal management of immunosuppressive treatment for these patients in order to obtain an immune state unfavorable to the activation of latent viruses, including EBV.

The Effect of Preemptive Rituximab Treatment in Patients with EBV Viremia after Solid Organ Transplantation on the Development of Post-Transplant Lymphoproliferative Disorder

The association between Epstein-Barr Virus (EBV) reactivation after solid organ transplantation (SOT) and Post-Transplant Lymphoproliferative Disorder (PTLD) is well described. There are currently no guidelines regarding the use of rituximab preemptively in patients with EBV viremia after SOT to reduce the risk of PTLD. We sought to determine whether rituximab in SOT patients with EBV viremia has benefit in preventing PTLD. This retrospective analysis of electronic medical record data identified SOT recipients who experienced EBV viremia post-transplant between January 2010 and February 2023 at Columbia University Irving Medical Center (CUIMC). Data queries were clinically reviewed. Logistic regression analysis was performed to investigate the relationship between predictor variables including rituximab administration, log-transformed EBV peak titer, duration of EBV viremia, and time from SOT to EBV reactivation, with the binary primary outcome of PTLD diagnosis status. The analysis encompassed both univariate and multivariate logistic regression. There were 2,168 patients who met the inclusion criteria of having EBV reactivation after SOT, out of 6,395 total SOT performed at CUIMC during this timeframe. Patient characteristics are described in table 1. The median peak EBV titer was 154 (range: not quantifiable - 5,335,816), the median duration of EBV viremia was 78 days (range: 1 - 6,980), and the median time from SOT to EBV reactivation was 688 days (range: 0-11103). Of the 2,168 patients, 185/2168 (8.5%) received rituximab for indications other than PTLD including EBV viremia, treatment of rejection, antibody desensitization therapy/immunosuppression peri-transplant, and other non-transplant related indications (e.g. lupus nephritis). Of the patients who received rituximab, 182/185 (98.4%) did not develop PTLD. The 3/185 (1.6%) patients who did develop PTLD were all ≤ 20 years-old at time of diagnosis, and had EBER(+), monomorphic PTLD, diffuse large B-cell lymphoma type, with a non-germinal center phenotype, involving the gastrointestinal tract. Two were CD20 positive, all 3 had partial weak CD30 positivity, and all 3 had clonal immunoglobulin heavy chain rearrangements. Among the patients who did not receive preemptive rituximab post-SOT, 195/1983 (9.8%) developed PTLD, whereas 1788/1983 (90.2%) did not. Prior rituximab therapy for any indication in patients with EBV viremia post-SOT was associated with lower risk of subsequent PTLD by univariate analysis (0.14% vs 8.39%, OR 0.15, p = 0.0013, 95% CI 0.048-0.48). Receipt of rituximab (OR 0.12, p=0.0002), log-transformed peak EBV titer (OR 1.10, p&amp;lt;0.0001), and duration of EBV viremia in months (OR 1.01, p&amp;lt;0.0001) were all independently associated with risk for PTLD by multivariate analysis (figure 1). The relationship between time from SOT to EBV viremia was not significantly associated with development of PTLD. This retrospective analysis suggests that administering preemptive rituximab in SOT patients with EBV viremia may lower the risk of developing PTLD. The fact that peak EBV titer and duration of EBV viremia remain associated with PTLD in multivariate analyses suggests that the benefit of rituximab may be independent of the degree or duration of EBV viremia. A prospective analysis of factors such as area under the curve of EBV exposure, HLA status, T cell clonotype diversity, organ type, and immunosuppression type, is needed to better understand individual risks for improved selection of those who would benefit from preemptive rituximab.

Umbilical Cord Blood Stem Cells As Third-Party Adjuvant Infusion in Human Leukocyte Antigen Antibody-Positive Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: The presence of human leukocyte antigen (HLA) antibodies, even if not donor-specific antibodies (DSA), is an important risk factor for graft failure (GF) or poor graft function (PGF) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, there is no ideal strategy for eradicating or descending HLA antibodies in this population. In the present study, we investigated the efficacy and safety of unrelated umbilical cord blood stem cells (CBCs) as a third-party adjuvant infusion in patients with HLA antibodies undergoing allo-HSCT. Methods:Patients undergoing allo-HSCT from July 2017 to June 2021 were enrolled in this study and HLA antibodies were routinely tested before transplantation. Patients were divided into three groups: Group A (patients with positive/weak positive HLA antibodies and with CBCs infusion), Group B (patients with HLA antibodies positive/weak positive without CBCs infusion), and Group C (patients without HLA antibody nor CBCs infusion). Single unrelated CBCs (HLA4/6 or more compatible ) were infused four hours before donor stem cells. The clinical characteristics of the patients and the paired donors were collected. The occurrence of granulocyte and platelet engraftment time, graft-versus-host disease (GVHD), cytomegalovirus (CMV), Epstein-Barr virus (EBV) reactivation, one-year and two-year relapse-free survival (RFS), transplantation-related mortality (TRM), and overall survival (OS) for all three groups of patients were also analyzed in this study. Results:A total of 90 patients were included in this study (17 in group A, 36 in group B, and 37 in group C). The baseline clinical characteristics of the patients were presented in Table 1. No obvious adverse effects were observed in the process of CBCs infusion. All the patients achieved 100% donor chimerism without CBCs engraftment. There were no significant differences in granulocyte and platelet engraftment time between the three groups. Two patients in Group A and C, and 3 patients in Group B developed PGF, respectively. There were no significant differences in the incidence and grades of acute or chronic GVHD among the three groups ( Figure A, B). In the first 3 months after allo-HSCT, the incidence of EBV reactivation was higher in patients in Group B than that in Group A and C, respectively ( Figure C). There were no significant differences in the CMV reactivation between the three groups in the first 3 months after transplantation ( Figure D). Patients in Group A and B both had lower 1-year RFS and higher 1-year TRM, compared with Group C, respectively ( Figure E, F). However, there were no significant statistical differences in 2-year RFS and TRM between the three groups. Although patients in Group A had longer 2-year OS than that in Group B, there were no significant differences between the two groups: Group A vs Group B (76.5% vs 55.6%, P = 0.194), Group B vs Group C (55.6% vs 85.5%, P &amp;lt; 0.05)( Figure G). A multifactorial Cox regression risk model was established to explore the risk factors affecting OS and suggested that patients' pre-transplantation status (with active malignant disease or minimal residual disease positive) and HLA antibodies pre-transplant were the most independent risk factors for OS (HR = 4.380, 95% CI = 1.544-12.427, P&amp;lt;0.05; HR = 0.196, 95% CI = 0.065-0.587, P&amp;lt;0.05). Conclusions: Patients with positive/weak positive HLA antibodies receiving CBCs as a third-party adjunctive infusion were safe, and there was a benefit in the incidence of EBV reactivation compared to such a population without CBCs infusion. The patients with negative HLA antibodies had higher 1-year RFS and lower 1-year TRM than those with positive/weakly positive HLA antibodies regardless CBCs infusion. Due to the small number of included cases, we will expand the sample number to further validate our results in the future.

Sitagliptin for Prevention of aGVHD in Patients Received Alternative Donor Transplantations: A Prospective, Multicenter, Open-Label, Randomized Controlled Trial

Background： Acute graft versus host disease (aGVHD) remains one of the most frequent complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with high mortality. Traditionally, alternative donor transplantation (ADT) from an unrelated donor or a haploidentical family donor has been reported in an increased incidence of aGVHD and severe GvHD. Sitagliptin is a selective inhibitor of dipeptidyl peptidase 4 (DPP-4; also known as CD26), a transmembrane receptor expressed on T cells which has a costimulatory function in activating T cells. This trial aims to evaluate the safety and efficacy of sitagliptin plus a calcineurin inhibitor, methotrexate (MTX), mycophenolate mofetil (MMF) and antithymocyte globulin (ATG) in the prevention of aGVHD for ADT. Methods： We are performing a prospective, multicenter, open-label, randomized controlled trial (NCT05149365). From 5 HSCT centers in China competitively, patients(pts) who underwent their first allo-HSCT in first or second complete remission (CR) state with hematologic malignancies were received busulfan and cyclophosphamide (Bu/Cy) myeloablative conditioning regimen followed by ADT. 190 eligible pts(18-60 years) were planned randomly assigned in a 1:1 ratio to either a sitagliptin group (600mg sitagliptin taken orally every 12 hours on day -1 to +14, plus conventional prophylaxis including Cyclosporin A(CsA)/ tacrolimus (FK506), MTX, MMF and ATG) and a conventional prophylaxis control group (CsA/FK506, MTX, MMF and ATG). The randomization was based on a computer-generated priori list of 190 binary numbers, with block-balanced with random variable block sizes of two or four pts, without stratification. The primary endpoint was grade II to IV aGvHD by day +100. Key secondary endpoints included severe (grade III or IV) aGvHD by day +100, transplant-related mortality (TRM), relapse-free survival (RFS) and GVHD-free, relapse-free survival (GRFS) from HSCT. At present we have completed pts enrollment and are at the stage of follow-up the study endpoints. Results : From December 2021 to May 2023, a total of 191 pts were enrolled. The ratio of male to female was 113 to 78 with a median age of 38 years (range, 18-60 years). Pts were randomly assigned to the sitagliptin group of 95 and the control group of 96. The clinical and transplantation characteristics at baseline of the intention to treat (ITT) population of the two groups were well balanced (Table1). The last follow-up was on May 30, 2023 and the median follow-up time was 164 (20-512) days. Comparison of the sitagliptin group and the control group by day +100, the cumulative incidence rate of grade II-IV aGVHD was 15.1% and 28.6%, respectively ( P=0.019), and the cumulative incidence rate of grade III or IV aGVHD was 7.6% and 15.9% ( P=0.068); median days of neutrophil engraftment were +11.48 and +11.65 days, respectively (P=0.545); TRM was 3.7% and 7.1% (P=0.454), RFS was 98.7% and 95.9% (P=0.889), respectively and OS of the two groups(96.7% vs 93.6%, P=0.341) was also the same as the RFS. There was no significant difference between the sitagliptin and control groups with regard to cytomegalovirus reactivation, Epstein-Barr virus reactivation, and transplant-related complications (Table 1, Figure 1). The regimen was well tolerated. Only 5(/95) pts discontinued sitagliptin treatment due to adverse events: 4 patients experienced abdominal pain (CTCAE Grade1-2) undergoing sitagliptin therapy 3-7 days and one of them also exhibited hypoglycemia (CTCAE Grade1), which were deemed by clinicians as probable (WHO-UMC) associated with sitagliptin. Symptoms resolved spontaneously within 2 days of cessation. Another patient discontinued treatment due to mild hematemesis initiating sitagliptin therapy only 2 days, which was determined by the clinician as unlikely (WHO-UMC) associated with sitagliptin. Conclusions : Our trial showed sitagliptin combined with conventional prophylaxis (CsA+MTX+MMF+ATG) resulted in a significant reduction of grade II-IV aGVHD in ADT. Furthermore, sitagliptin is ready available, easy of administration, safe, and relatively inexpensive. It remains to be followed up whether it would increase infection and relapse or improve the prognosis of patients after allo-HSCT.

Addition of Daclizumab to Standard Graft-Versus-Host Disease Prophylaxis in Patients with Thalassemia Major Undergoing Transplantation from Related Haploidentical Donor and Unrelated Donor Hematopoietic Stem-Cell Transplantation: A Single-Center Retrospective Analysis from China

Background: Previous studies have confirmed that the incidence of graft-versus-host disease (GVHD) remained still very high in patients with thalassemia major (TM) undergoing related haploidentical donor and unrelated donor hematopoietic stem-cell transplantation (HID-HSCT and MUD-HSCT), which seriously affected patients' overall survival and thalassemia-free survival, especially acute GVHD (aGVHD). This study aimed to investigate whether Daclizumab at a dose of 1 mg/Kg on days +7, +14, +28 and +42 reduced GVHD plus standard GVHD prophylaxis (tacrolimus [FK506] plus methotrexate [MTX] and mycophenolate mofetil [MMF]) in TM patients with HID-HSCT or MUD-HSCT. Methods: We retrospectively analyzed our data of TM patients who underwent HID-HSCT or MUD-HSCT at the First Affiliated Hospital of Guangxi Medical University in China from November 2022 to July 2023. TM patients enrolled all received the GVHD prophylaxis which included Daclizumab at a dose of 1 mg/kg on days +7, +14, +28 and +42 plus FK506 (0.03mg/kg/day), MTX (15mg/m 2 on days +1, 10mg/m 2 on days +3, +6, +11), and MMF (250mg/day × 90 days). The primary endpoint was grades 2-4 aGVHD on days +100. Results: Data of 37 patients' transplantation were evaluated. The ratio of male to female was 20 to 17 with a median age of 8 years (range, 3-17 years). Patients were assigned to the HID-HSCT of 24 and the MUD-HSCT of 13. Median follow-up period was 7 months (range, 3 to 11 months). The total of the cumulative incidence rate of grades 2-4 aGVHD was 18.9%, and the rate of grades 3-4 aGVHD was 10.8%. Subgroup analysis showed that the rate of grades 2-4 aGVHD was 20.8% in HID-HSCT and 15.4% in MUD-HSCT, respectively; the rate of grades 3-4 aGVHD was 8.3% in HID-HSCT and 15.4% in MUD-HSCT, respectively. The median days of neutrophil engraftment were +12 days, platelets Implantation days were +14 days. The cytomegalovirus reactivation, Epstein-Barr virus reactivation, fungal infection and septicemia were seen in 16(43.2%), 3(8.1%), 6(16.2%) and 5(13.5%) patients, respectively. One patient in MUD-HSCT died of severe pulmonary infection, but no deaths were attributable to HID-HSCT (Figure 1, Table 1). CONCLUSION: The results of our study analysis suggest that Daclizumab at a dose of 1 mg/kg on days +7, +14, +28 and +42 provides clinically meaningful benefits when added to standard GVHD prophylaxis in TM patients undergoing haploidentical donor and unrelated donor transplantation, including decrease in the incidence rate of aGVHD without sever infection and transplant-related complications. Four doses of Daclizumab should be included in the GVHD prophylaxis of patients with thalassemia major selected for related haploidentical donor and unrelated donor transplantation.

A Co-Reactivation Model of Cytomegalovirus and Epstein-Barr Virus after Allogeneic Hematopoietic Stem Cell Transplantation Was Constructed to Predict Mortality after Transplantation

Abstract Objective: The aim of this study was to create a predictive model for the occurrence of co-reactivation of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: The study included 373 patients who underwent allo-HSCT. The patient population was randomly divided into two groups: a training cohort comprising 70% of patients (n=261) and a validation cohort comprising 30% of patients (n=112). The predictive model was developed using several variables, including the time of reactivation of CMV and EBV, patient age and gender, presence of hepatitis B core antibody (HbcAb), type of transplant, use of anti-thymocyte globulin (ATG), and the occurrence of acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively). Results: The developed predictive model was expressed as y = -6.21338339 + 0.01923832 × (CMV reactivation time) + 0.00362628 × (EBV reactivation time) - 0.57604523 × (patient gender) + 0.01974136 × (patient age) + 0.85514424 × (presence of HbcAb) + 1.13782872 × (transplant type) + 0.95376138 × (use of ATG) - 0.62393602 × (aGVHD) - 1.09485262 × (cGVHD). The model demonstrated a g-mean value of 0.685 and a probability threshold of 0.297, allowing for the classification of patients into high-risk and low-risk groups. The incidence of co-reactivation of CMV and EBV over 100 days was significantly higher in the high-risk group compared to the low-risk group. In the high-risk group, the incidence was 49.3%, while in the low-risk group, it was 16.9% (P&amp;lt;0.0001, HR 95%CI 3.693(2.493,5.47)). For patients with aGVHD, the incidence was 41.7% in the high-risk group and 17.9% in the low-risk group (P=0.00026, HR 95%CI 2.7473(1.560,4.839)). In patients without aGVHD, the incidence was 53% in the high-risk group and 14.0% in the low-risk group (P&amp;lt;0.0001, HR 95%CI 4.9439(2.347,10.4)). Additionally, the model was able to predict post-transplant mortality and survival. Conclusion: Overall, the findings of this study suggest that the developed model is a valuable tool for predicting the risk of CMV and EBV co-reactivation following allo-HSCT. By identifying high-risk patients, clinicians can take proactive measures to prevent or manage viral infections, ultimately improving patient outcomes. Therefore, this model can potentially enhance clinical decision-making and optimize post-transplant care.