TianTan vaccinia virus-based EBV vaccines targeting both latent and lytic antigens elicits potent immunity against lethal EBV challenge in humanized mice.

Epstein-Barr virus (EBV) infection has been related to multiple epithelial cancers and lymphomas. Current efforts in developing a prophylactic EBV vaccine have focused on inducing neutralizing antibodies. However, given the lifelong and persistent nature of EBV infection following primary infection, it is rationalized that an ideal vaccine should elicit both humoral and cellular immune responses targeting multiple stages of the EBV lifecycle. This study used a DNA vector and a TianTan vaccinia virus to express key EBV antigens, including BZLF1, EBNA1, EBNA3B, and gH/gL, to generate multi-antigen vaccines. The multi-antigen vaccine expressing all four antigens and the multi-antigen vaccine expressing BZLF1, EBNA1, and EBNA3B showed comparable protection effects and prevented 100% and 80% of humanized mice, respectively, from EBV-induced fatal B cell lymphoma by activating BZLF1, EBNA1, and EBNA3B specific T cell. The vaccine expressing lytic protein BZLF1 elicited stronger T cell responses and conferred superior protection compared to vaccines targeting single latent EBNA1 or EBNA3B. The vaccine solely expressing gH/gL exhibited no T cell protective effects in our humanized mice model. Our study implicates the potential of EBV vaccines that induce potent cellular responses targeting both latent and lytic phases of the EBV life cycle in the prevention of EBV-induced B cell lymphoma.

Increased Epstein‒Barr virus reactivation following prophylaxis for cytomegalovirus infection after haploidentical haematopoietic stem cell transplantation.

Letermovir (LTV) prophylaxis is effective in reducing the incidence of clinically significant cytomegalovirus (CMV) infection (cs CMVi) after allogeneic haematopoietic stem cell transplantation (allo-HSCT). Since our centre began administering LTV prophylaxis in June 2022, we have observed a certain increase in the incidence of Epstein-Barr virus (EBV) reactivation after haploidentical HSCT. We retrospectively analysed 230 consecutive patients who underwent haploidentical HSCT with rabbit anti-thymocyte globulin (ATG) from October 2022 to June 2023. The LTV group included 133 patients who received LTV prophylaxis, and the control group included 97 patients who did not receive LTV prophylaxis. At 1 year after HSCT, EBV reactivation was observed in 36 patients (27%) in the LTV group and 13 patients (13%) in the control group (p = 0.012). All patients with EBV reactivation had EBV-DNAemia, and one patient in each group developed EBV-associated posttransplantation lymphoproliferative disorder (PTLD). The proportion of patients with low EBV-DNA loads (> 5 × 102 to < 1 × 104 copies/mL) was greater in the LTV group than in the control group (23% vs. 10%, p = 0.01). The proportion of patients with CMV reactivation was lower in the LTV group than in the control group (35% vs. 56%, p = 0.002). There was no significant difference between the groups in terms of neutrophil and platelet count recovery, the cumulative incidence of acute/chronic graft-versus-host disease, overall survival, cumulative relapse rate or nonrelapse mortality. Our results show that the increased incidence of EBV reactivation may be associated with LTV prophylaxis for CMV after haploidentical HSCT.

Multivalent MVA-vectored vaccine elicits EBV neutralizing antibodies in rhesus macaques that reduce EBV infection in humanized mice.

Introduction: Epstein-Barr virus (EBV) is an oncogenic human herpesvirus associated with ~350,000 cases of lymphoid and epithelial malignancies every year, and is etiologically linked to infectious mononucleosis and multiple sclerosis. Despite four decades of research, no EBV vaccine candidate has yet reached licensure. Most previous vaccine attempts focused on a single viral entry glycoprotein, gp350, but recent data from clinical and pre-clinical studies, and the elucidation of viral entry mechanisms, support the inclusion of multiple entry glycoproteins in EBV vaccine design. Methods: Here we generated a modified vaccinia Ankara (MVA)-vectored EBV vaccine, MVA-EBV5-2, that targets five EBV entry glycoproteins, gp350, gB, and the gp42gHgL complex. We characterized the genetic and translational stability of the vaccine, followed by immunogenicity assessment in BALB/c mice and rhesus lymphocryptovirus-negative rhesus macaques as compared to a gp350-based MVA vaccine. Finally, we assessed the efficacy of MVA-EBV5-2-immune rhesus serum at preventing EBV infection in human CD34+ hematopoietic stem cell-reconstituted NSG mice, under two EBV challenge doses. Results: The MVA-EBV5-2 vaccine was genetically and translationally stable over 10 viral passages as shown by genetic and protein expression analysis, and when administered to female and male BALB/c mice, elicited serum EBV-specific IgG of both IgG1 and IgG2a subtypes with neutralizing activity in vitro. In Raji B cells, this neutralizing activity outperformed that of serum from mice immunized with a monovalent MVA-vectored gp350 vaccine. Similarly, MVA-EBV5-2 elicited EBV-specific IgG in rhesus macaques that were detected in both serum and saliva of immunized animals, with serum antibodies demonstrating neutralizing activity in vitro that outperformed serum from MVA-gp350-immunized macaques. Finally, pre-treatment with serum from MVA-EBV5-2-immunized macaques resulted in fewer EBV-infected mice in the two challenge experiments than pretreatment with serum from pre-immune macaques or macaques immunized with the monovalent gp350-based vaccine. Discussion: These results support the inclusion of multiple entry glycoproteins in EBV vaccine design and position our vaccine as a strong candidate for clinical translation.

Impact of prophylaxis with rituximab on EBV-related complications after allogeneic hematopoietic cell transplantation in children.

Background: Children undergoing allo-HCT are at high risk of EBV-related complications. The objective of the study was to analyze the impact of prophylactic post-transplant rituximab on EBV infection and EBV-PTLD in children after allo-HCT, to determine the risk factors for the development of EBV infection and EBV-PTLD and to determine their outcomes. Additionally, the impact of EBV-driven complications on transplant outcomes was analyzed. Methods: Single center retrospective analysis of EBV-related complications in pediatric population undergoing allo-HCT, based on strategy of prophylaxis with rituximab. Overall 276 consecutive children, including 122 on prophylaxis, were analyzed for EBV-driven complications and transplant outcomes. Results: Prophylaxis with rituximab resulted in significant reduction of EBV infection (from 35.1% to 20.5%; HR=2.7; p<0.0001), and EBV-PTLD (from 13.0% to 3.3%; HR=0.23; p=0.0045). A trend for improved survival was also observed (HR=0.66; p=0.068), while non-relapse mortality was comparable in both cohorts. The peak value of viral load was a risk factor in the development of EBV-PTLD: 10-fold higher peak viral load in comparison to the baseline 104 copies/mL, caused a 3-fold (HR=3.36; p<0.001) increase in the risk of EBV-PTLD. Rituximab treatment was effective as a preemptive therapy in 91.1%, and in 70.9% in EBV-PTLD. Patients who developed PTLD had dismal 5-year overall survival (29% vs 60%; p<0.001), and an increased risk of relapse (72% vs 35%; p=0.024). Conclusions: Rituximab for prophylaxis of EBV infection and EBV-PTLD was highly effective in pediatric population. Treatment of EBV-PTLD was successful in 70%, however the occurrence of EBV-PTLD was associated with an increased risk of relapse of primary malignant disease.

Exploring the nuclear proteins, viral capsid protein, and early antigen protein using immunoinformatic and molecular modeling approaches to design a vaccine candidate against Epstein Barr virus.

The available Epstein Barr virus vaccine has tirelessly harnessed the gp350 glycoprotein as its target epitope, but the result has not been preventive. Right here, we designed a global multi-epitope vaccine for EBV; with special attention to making sure all strains and preventive antigens are covered. Using a robust computational vaccine design approach, our proposed vaccine is armed with 6-16 mers linear B-cell epitopes, 4-9 mer CTL epitopes, and 8-15 mer HTL epitopes which are verified to induce interleukin 4, 10 & IFN-gamma. We employed deep computational mining coupled with expert intelligence in designing the vaccine, using human Beta defensin-3-which has been reported to induce the same TLRs as EBV-as the adjuvant. The tendency of the vaccine to cause autoimmune disorder is quenched by the assurance that the construct contains no EBNA-1 homolog. The protein vaccine construct exhibited excellent physicochemical attributes such as Aliphatic index 59.55 and GRAVY - 0.710; and a ProsaWeb Z score of - 3.04. Further computational analysis revealed the vaccine docked favorably with EBV indicted TLR 1, 2, 4 & 9 with satisfactory interaction patterns. With global coverage of 85.75% and the stable molecular dynamics result obtained for the best two interactions, we are optimistic that our nontoxic, non-allergenic multi-epitope vaccine will help to ameliorate the EBV-associated diseases-which include various malignancies, tumors, and cancers-preventively.

GB and gH/gL fusion machinery: a promising target for vaccines to prevent Epstein-Barr virus infection.

Epstein‒Barr virus (EBV) is a double-stranded DNA virus belonging to the family Orthoherpesviridae that is associated with the development of various tumors, such as lymphoma, nasopharyngeal carcinoma, and gastric cancer. There are no uniformly effective treatments for human EBV infection, and vaccines and immunotherapies are currently the main research directions. The glycoproteins gB and gH/gL are surface glycoproteins that are common to all herpesviruses, with subtle differences in structure and function between different viruses. The core membrane fusion machinery constituted by EBV gB and gH/gL is an important target of neutralizing antibodies in epithelial EBV infection due to its essential role in the fusion of viral and target cell membranes. In this article, we review the main modes of EBV infection, the structure and function of the core fusion machinery gB and gH/gL, and the development of neutralizing antibodies and prophylactic vaccines based on this target.

A cocktail nanovaccine targeting key entry glycoproteins elicits high neutralizing antibody levels against EBV infection.

Epstein-Barr virus (EBV) infects more than 95% of adults worldwide and is closely associated with various malignancies. Considering the complex life cycle of EBV, developing vaccines targeting key entry glycoproteins to elicit robust and durable adaptive immune responses may provide better protection. EBV gHgL-, gB- and gp42-specific antibodies in healthy EBV carriers contributed to sera neutralizing abilities in vitro, indicating that they are potential antigen candidates. To enhance the immunogenicity of these antigens, we formulate three nanovaccines by co-delivering molecular adjuvants (CpG and MPLA) and antigens (gHgL, gB or gp42). These nanovaccines induce robust humoral and cellular responses through efficient activation of dendritic cells and germinal center response. Importantly, these nanovaccines generate high levels of neutralizing antibodies recognizing vulnerable sites of all three antigens. IgGs induced by a cocktail vaccine containing three nanovaccines confer superior protection from lethal EBV challenge in female humanized mice compared to IgG elicited by individual NP-gHgL, NP-gB and NP-gp42. Importantly, serum antibodies elicited by cocktail nanovaccine immunization confer durable protection against EBV-associated lymphoma. Overall, the cocktail nanovaccine shows robust immunogenicity and is a promising candidate for further clinical trials.

Vaccination with nanoparticles displaying gH/gL from Epstein-Barr virus elicits limited cross-protection against rhesus lymphocryptovirus.

Epstein-Barr virus (EBV) is associated with infectious mononucleosis, cancer, and multiple sclerosis. A vaccine that prevents infection and/or EBV-associated morbidity is an unmet need. The viral gH/gL glycoprotein complex is essential for infectivity, making it an attractive vaccine target. Here, we evaluate the immunogenicity of a gH/gL nanoparticle vaccine adjuvanted with the Sigma Adjuvant System (SAS) or a saponin/monophosphoryl lipid A nanoparticle (SMNP) in rhesus macaques. Formulation with SMNP elicits higher titers of neutralizing antibodies and more vaccine-specific CD4+ T cells. All but one animal in the SMNP group were infected after oral challenge with the EBV ortholog rhesus lymphocryptovirus (rhLCV). Their immune plasma had a 10- to 100-fold lower reactivity against rhLCV gH/gL compared to EBV gH/gL. Anti-EBV neutralizing monoclonal antibodies showed reduced binding to rhLCV gH/gL, demonstrating that EBV gH/gL neutralizing epitopes are poorly conserved on rhLCV gH/gL. Prevention of rhLCV infection despite antigenic disparity supports clinical development of gH/gL nanoparticle vaccines against EBV.

Effect of pre-emptive rituximab on EBV DNA levels and prevention of post-transplant lymphoproliferative disorder in pediatric kidney transplant recipients: A case series from the pediatric nephrology research consortium.

BACKGROUND: There are scant data on the effect of rituximab on EBV DNA levels and prevention of post-transplant lymphoproliferative disorder (PTLD) in pediatric kidney transplant recipients with EBV DNAemia. METHODS: Kidney transplant recipients with EBV DNAemia treated with rituximab to prevent PTLD between 7/1999 and 7/2019 at five pediatric centers were included. Those with confirmed PTLD at the onset of rituximab were excluded. Primary outcomes included percentage change in EBV DNAemia and occurrence of PTLD post rituximab. RESULTS: Twenty-six pediatric kidney transplant recipients were included. Median age at transplant was 4 years (IQR 2.1-10.3). EBV DNA load monitoring by qPCR was performed at 1-3 month intervals. EBV DNAemia onset occurred at a median of 73 days post-transplant (IQR 52-307), followed by DNAemia peak at a median of 268 days (IQR 112-536). Rituximab was administered at a median of 9 days post peak (IQR 0-118). Rituximab regimens varied; median dose 375 mg/m2 (IQR 375-439) weekly for 1-4 doses per course. Following rituximab, EBV DNA load decreased to <10% of baseline at 120 days in 20/26 patients; however, only 30% achieved complete resolution at last follow-up (median 2094 days post-transplant [IQR 1538-3463]). Two (7%) developed PTLD at 915 and 1713 days post rituximab. All recipients had functioning grafts. One death occurred in a child with PTLD following remission due to unrelated reasons. CONCLUSIONS: In the largest pediatric kidney transplant recipient case series with EBV DNAemia given rituximab to prevent PTLD, rituximab achieved a short-term reduction in DNA load; however, recurrent DNAemia is common.

A Focused Review of Epstein-Barr Virus Infections and PTLD in Pediatric Transplant Recipients: Guidance From the IPTA and ECIL Guidelines.

Epstein-Barr Virus (EBV) diseases, including EBV-associated post-transplant lymphoproliferative disorder (PTLD) remain important causes of morbidity and mortality in children undergoing solid organ transplantation (SOT) and hematopoietic cell transplantation (HCT). Despite progress in the prevention of EBV disease including PTLD (EBV/PTLD) in HCT, key questions in the prevention, and management of these infectious complications remain unanswered. The goal of this manuscript is to highlight key points and recommendations derived from the consensus guidelines published by the International Pediatric Transplant Association and the European Conference on Infections in Leukemia for children undergoing SOT and HCT, respectively. Additionally, we provide background and guidance on the use of EBV viral load measurement in the prevention and management of these children.

Rituximab for posttransplant lymphoproliferative disorder - therapeutic, preemptive, or prophylactic?

To minimize mortality due to posttransplant lymphoproliferative disorder (PTLD), the following strategies have been used: (1) Therapy without EBV Monitoring, i.e., administration of rituximab after PTLD diagnosis, usually by biopsy, in the absence of routine Epstein-Barr virus (EBV) DNAemia monitoring, (2) Prompt Therapy, i.e., monitoring EBV DNAemia, searching for PTLD by imaging when the DNAemia has exceeded a pre-specified threshold, and administration of rituximab if the imaging is consistent with PTLD, (3) Preemptive Therapy, i.e., monitoring EBV DNAemia and administration of rituximab when the DNAemia has exceeded a pre-specified threshold, and (4) Prophylaxis, i.e., administration of rituximab to all transplant recipients. The superiority of one of these strategies over the other strategies has not been established. Here we review the pros and cons of each strategy. Preemptive therapy or prophylaxis may currently be preferred for patients who are at a high risk of dying due to PTLD. However, Therapy without EBV Monitoring may be used for both high- and low-risk patients in the future, if effective and relatively non-toxic therapies for rituximab-refractory PTLD (e.g., EBV-specific T cells) have become easily available.

Exploring the cost-effectiveness of EBV vaccination to prevent multiple sclerosis in an Australian setting.

BACKGROUND: Increasing evidence suggests the potential of Epstein-Barr virus (EBV) vaccination in preventing multiple sclerosis (MS). We aimed to explore the cost-effectiveness of a hypothetical EBV vaccination to prevent MS in an Australian setting. METHODS: A five-state Markov model was developed to simulate the incidence and subsequent progression of MS in a general Australian population. The model inputs were derived from published Australian sources. Hypothetical vaccination costs, efficacy and strategies were derived from literature. Total lifetime costs, quality-adjusted life years (QALYs) and incremental cost-effectiveness ratios (ICERs) were estimated for two hypothetical prevention strategies versus no prevention from the societal and health system payer perspectives. Costs and QALYs were discounted at 5% annually. One-way, two-way and probabilistic sensitivity analyses were performed. RESULTS: From societal perspective, EBV vaccination targeted at aged 0 and aged 12 both dominated no prevention (ie, cost saving and increasing QALYs). However, vaccinating at age 12 was more cost-effective (total lifetime costs reduced by $A452/person, QALYs gained=0.007, ICER=-$A64 571/QALY gained) than vaccinating at age 0 (total lifetime costs reduced by $A40/person, QALYs gained=0.003, ICER=-$A13 333/QALY gained). The probabilities of being cost-effective under $A50 000/QALY gained threshold for vaccinating at ages 0 and 12 were 66% and 90%, respectively. From health system payer perspective, the EBV vaccination was cost-effective at age 12 only. Sensitivity analyses demonstrated the cost-effectiveness of EBV vaccination to prevent MS under a wide range of plausible scenarios. CONCLUSIONS: MS prevention using future EBV vaccinations, particularly targeted at adolescence population, is highly likely to be cost-effective.

mRNA vaccines in the rheumatologist's future.

BACKGROUND: Vaccines taking advantage of mRNA technology have been long in development. OBJECTIVES: To review the status of approved mRNA vaccines for infectious diseases as well as those in development. METHODS: Systematic literature review of clinical and immunologic studies of mRNA vaccines against infectious diseases. RESULTS: Currently approved mRNA vaccines include those against SARS CoV-2 virus. They are immunogenic and provide good protection against severe disease. A number of mRNA vaccines for influenza are in development including in phase 3 studies. Other such vaccines in development include those targeting Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), varicella (VZV), and respiratory syncytial virus (RSV). Many of these vaccines will likely be indicated for immunosuppressed populations including those with autoimmune inflammatory diseases. CONCLUSIONS: A number of mRNA vaccines against viral pathogens are in early to late phase development. The ability to rapidly change vaccine constituents and target complex targets, make mRNA vaccines likely to be adopted in the future.

A gB nanoparticle vaccine elicits a protective neutralizing antibody response against EBV.

Epstein-Barr virus (EBV) is a global public health concern, as it is known to cause multiple diseases while also being etiologically associated with a wide range of epithelial and lymphoid malignancies. Currently, there is no available prophylactic vaccine against EBV. gB is the EBV fusion protein that mediates viral membrane fusion and participates in host recognition, making it critical for EBV infection in both B cells and epithelial cells. Here, we present a gB nanoparticle, gB-I53-50 NP, that displays multiple copies of gB. Compared with the gB trimer, gB-I53-50 NP shows improved structural integrity and stability, as well as enhanced immunogenicity in mice and non-human primate (NHP) preclinical models. Immunization and passive transfer demonstrate a robust and durable protective antibody response that protects humanized mice against lethal EBV challenge. This vaccine candidate demonstrates significant potential in preventing EBV infection, providing a possible platform for developing prophylactic vaccines for EBV.

Lymph node targeted multi-epitope subunit vaccine promotes effective immunity to EBV in HLA-expressing mice.

The recent emergence of a causal link between Epstein-Barr virus (EBV) and multiple sclerosis has generated considerable interest in the development of an effective vaccine against EBV. Here we describe a vaccine formulation based on a lymph node targeting Amphiphile vaccine adjuvant, Amphiphile-CpG, admixed with EBV gp350 glycoprotein and an engineered EBV polyepitope protein that includes 20 CD8+ T cell epitopes from EBV latent and lytic antigens. Potent gp350-specific IgG responses are induced in mice with titers >100,000 in Amphiphile-CpG vaccinated mice. Immunization including Amphiphile-CpG also induces high frequencies of polyfunctional gp350-specific CD4+ T cells and EBV-specific CD8+ T cells that are 2-fold greater than soluble CpG and are maintained for >7 months post immunization. This combination of broad humoral and cellular immunity against multiple viral determinants is likely to provide better protection against primary infection and control of latently infected B cells leading to protection against the development of EBV-associated diseases.

Mendelian randomisation identifies priority groups for prophylactic EBV vaccination.

BACKGROUND: Epstein Barr virus (EBV) infects ~ 95% of the population worldwide and is known to cause adverse health outcomes such as Hodgkin's, non-Hodgkin's lymphomas, and multiple sclerosis. There is substantial interest and investment in developing infection-preventing vaccines for EBV. To effectively deploy such vaccines, it is vital that we understand the risk factors for infection. Why particular individuals do not become infected is currently unknown. The current literature, describes complex, often conflicting webs of intersecting factors-sociodemographic, clinical, genetic, environmental-, rendering causality difficult to decipher. We aimed to use Mendelian randomization (MR) to overcome the issues posed by confounding and reverse causality to determine the causal risk factors for the acquisition of EBV. METHODS: We mapped the complex evidence from the literature prior to this study factors associated with EBV serostatus (as a proxy for infection) into a causal diagram to determine putative risk factors for our study. Using data from the UK Biobank of 8422 individuals genomically deemed to be of white British ancestry between the ages of 40 and 69 at recruitment between the years 2006 and 2010, we performed a genome wide association study (GWAS) of EBV serostatus, followed by a Two Sample MR to determine which putative risk factors were causal. RESULTS: Our GWAS identified two novel loci associated with EBV serostatus. In MR analyses, we confirmed shorter time in education, an increase in number of sexual partners, and a lower age of smoking commencement, to be causal risk factors for EBV serostatus. CONCLUSIONS: Given the current interest and likelihood of a future EBV vaccine, these factors can inform vaccine development and deployment strategies by completing the puzzle of causality. Knowing these risk factors allows identification of those most likely to acquire EBV, giving insight into what age to vaccinate and who to prioritise when a vaccine is introduced.

How I prevent viral reactivation in high-risk patients.

Preventing viral infections at an early stage is a key strategy for successfully improving transplant outcomes. Preemptive therapy and prophylaxis with antiviral agents have been successfully used to prevent clinically significant viral infections in hematopoietic cell transplant recipients. Major progress has been made over the past decades in preventing viral infections through a better understanding of the biology and risk factors, as well as the introduction of novel antiviral agents and advances in immunotherapy. High-quality evidence exists for the effective prevention of herpes simplex virus, varicella-zoster virus, and cytomegalovirus infection and disease. Few data are available on the effective prevention of human herpesvirus 6, Epstein-Barr virus, adenovirus, and BK virus infections. To highlight the spectrum of clinical practice, here we review high-risk situations that we handle with a high degree of uniformity and cases that feature differences in approaches, reflecting distinct hematopoietic cell transplant practices, such as ex vivo T-cell depletion.

Pre-Hematopoietic Stem Cell Transplantation Rituximab for Epstein-Barr Virus and Post-Lymphoproliferative Disorder Prophylaxis in Alemtuzumab Recipients.

Epstein-Barr virus (EBV) reactivation and EBV-related post-transplantation lymphoproliferative disorder (PTLD) are often fatal complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The risk of EBV reactivation may be mitigated by depletion of B cells with rituximab. Starting in January 2020, allo-HSCT recipients undergoing T-cell depletion with alemtuzumab received 1 dose of rituximab before transplantation. The objective of this study was to evaluate the cumulative incidence of EBV reactivation and EBV-PTLD in recipients of allo-HSCT and in vivo T-cell depletion with alemtuzumab who received pre-HSCT rituximab compared to patients who did not. This was a single-center retrospective analysis of adult patients who consecutively received an HLA-identical allo-HSCT between January 2019 and May 2021 and in vivo T-cell depletion with alemtuzumab. Patients were included in the rituximab cohort if they received rituximab within 6 months before their transplantation. The primary endpoint was incidence of EBV reactivation at day 180 among those receiving pre-HSCT rituximab versus those not receiving rituximab. Secondary endpoints included cumulative incidence of EBV-PTLD at 1 year, time to engraftment, immune reconstitution, and incidence of infections and acute graft-versus-host disease (aGVHD) at day 180. Eighty-six consecutive patients who received an allo-HSCT with alemtuzumab T-cell depletion were reviewed; 43 patients who received pre-HSCT rituximab after our protocol modification were compared to 43 patients who did not receive pre-HSCT rituximab before this change. Median age was 57 (interquartile range [IQR] 40-69) years, and the majority of patients had acute myeloid leukemia or myelodysplastic syndrome. Baseline characteristics were similar between the cohorts. EBV reactivation at day 180 occurred in 23 (53%) patients without prior rituximab exposure versus 0 patients with pre-HSCT rituximab exposure (P < .0001). Similarly, 6 patients without prior rituximab exposure developed PTLD at 1 year compared to no cases of PTLD among patients receiving pre-HSCT rituximab. There was no difference in neutrophil engraftment, incidence of infections, or aGVHD at day 180 between the 2 cohorts. There was a delay in time to platelet engraftment in the rituximab cohort (median 16 [IQR 15-20] days versus 15 [IQR 14-17] days; P = .04). Administration of pre-HSCT rituximab before allo-HSCT in patients receiving T-cell depletion with alemtuzumab was associated with a significant decrease in the risk for EBV reactivation and EBV-PTLD, without increasing aGVHD or infection rates.