CMV-specific Immune Response Research Articles

Cytomegalovirus UL44 protein induces a potent T-cell immune response in mice

Due to the severity of CMV infection in immunocompromised individuals the development of a vaccine has been declared a priority. However, despite the efforts made there is no yet a vaccine available for clinical use. We designed an approach to identify new CMV antigens able to inducing a broad immune response that could be used in future vaccine formulations.We have used serum samples from 28 kidney transplant recipients, with a previously acquired CMV-specific immune response to identify viral proteins that were recognized by the antibodies present in the patient serum samples by Western blot. A band of approximately 45 kDa, identified as UL44, was detected by most serum samples. UL44 immunogenicity was tested in BALB/c mice that received three doses of the UL44-pcDNA DNA vaccine. UL44 elicited both, a strong antibody response and CMV-specific cellular response. Using bioinformatic analysis we demonstrated that UL44 is a highly conserved protein and contains epitopes that are able to activate CD8 lymphocytes of the most common HLA alleles in the world population. We constructed a UL44 ORF deletion mutant virus that produced no viral progeny, suggesting that UL44 is an essential viral protein. In addition, other authors have demonstrated that UL44 is one of the most abundant viral proteins after infection and have suggested an essential role of UL44 in viral replication. Altogether, our data suggests that UL44 is a potent antigen, and favored by its abundance, it may be a good candidate to include in a vaccine formulation.

Immunomonitoring via ELISPOT Assay Reveals Attenuated T-Cell Immunity to CMV in Immunocompromised Liver-Transplant Patients.

Assessing immune responses to cytomegalovirus (CMV) after liver transplant in patients on immunosuppressive therapy remains challenging. In this study, employing ELISPOT assays, 52 liver-transplant recipients were evaluated for antiviral T-cell activity in peripheral blood mononuclear cells (PBMCs), measuring interferon-γ (IFN-γ) secretion upon stimulation with CMV-specific peptides (CMV peptide pool, CMV IE-1, and pp65 antigens). Parameters such as stimulation index, mean spot size, and mean spot count were measured. The study found that heightened immunosuppression, especially with prednisolone in triple therapy, significantly dampened CMV-specific immune responses. This was demonstrated by decreased IFN-γ production by CMV-specific T-cells (CMV peptide pool: p = 0.036; OR = 0.065 [95% CI: 0.005-0.840], pp65 antigen: p = 0.026; OR = 0.048 [95% CI: 0.003-0.699]). Increased immunosuppression correlated with reduced IFN-γ secretion per cell, reflected in smaller mean spot sizes for the CMV peptide pool (p = 0.019). Notably, shorter post-transplant intervals correlated with diminished antiviral T-cell IFN-γ release at two years (CMV peptide pool: p = 0.019; IE antigen: p = 0.010) and five years (CMV peptide pool: p = 0.0001; IE antigen: p = 0.002; pp65 antigen: p = 0.047), as did advancing age (pp65 antigen: p = 0.016, OR = 0.932, 95% CI: 0.881-0.987). Patients with undetectable CMV antigens had a notably higher risk of CMV reactivation within six months from blood collection, closely linked with triple immunosuppression and prednisolone use. These findings highlight the intricate interplay between immunosuppression, immune response dynamics, and CMV reactivation risk, emphasizing the necessity for tailored immunosuppressive strategies to mitigate CMV reactivation in liver-transplant recipients. It can be concluded that, particularly in the early months post-transplantation, the use of prednisolone as a third immunosuppressant should be critically reconsidered. Additionally, the use of prophylactic antiviral therapy effective against CMV in this context holds significant importance.

191 CMV-specific adaptive immune response in U.S. Mennonite patients with hypomorphic RAG1 or RAG2 mutations presenting with clinical variability

191 CMV-specific adaptive immune response in U.S. Mennonite patients with hypomorphic RAG1 or RAG2 mutations presenting with clinical variability

Dynamics of Immune Reconstitution after Unrelated Allogeneic Transplantation with Post-Transplantation Cyclophosphamide Compared to Classical Immunosuppression with ATG

Introduction: Post-transplant cyclophosphamide (PTCY), in hematopoietic stem cell transplantation (HSCT), is responsible for the success of haploidentical stem cell transplantation and is also being used in matched transplants such as unrelated transplants, with promising outcomes in graft versus host disease (GVHD). However, we lack data on immune reconstitution with this type of immunosuppression, especially in unrelated transplantation. Immune reconstitution is directly involved with post-transplant morbidity and mortality, being related to GVHD, relapses and infections, such as cytomegalovirus (CMV). Objectives: To evaluate global and CMV-specific immune reconstitution after unrelated donor HSCT, comparing the use of thymoglobulin (ATG) or PTCY protocol. Methods: We prospectively included patients undergoing first unrelated donor HSCT with ATG, methotrexate and tacrolimus or PTCY (50mg/Kg D+3 and D+4), mycophenolate mofetil (MMF) and tacrolimus with patient and/or donor CMV IgG positive. Immune reconstitution analyzes were performed by flow cytometry and CMV-specific recovery by ELISPot technique on days +30, +60, +90 and +180 post HSCT. Results: Thirty-six patients were included in this study, 20 in the ATG group and 16 in the PTCY group (table 1). Only 2 patients were 9x10 match, and the others were 10x10 match. All patients were submitted to preemptive strategy of CMV treatment, without the use of letermovir. Regarding clinical outcomes, neutrophilic engraftment was earlier with ATG (median of 12 days), compared to PTCY (median of 16 days) (p=0.0001). There was no difference between the groups in terms of length of hospital stay (median 33 days in both groups), CMV reactivation (70% in ATG and 75% PTCY, p=0.98), acute GVHD grades II to IV (p=0.26) or grades III-IV (p=0.56), and chronic GVHD (p=0.45), as well as relapse (p=0.3), non relapse mortality (p=0.77) and overall survival (p=0.4). Regarding immune reconstitution we observed an initial expansion of monocytes and NK lymphocytes in both groups and a higher increase in CD8+ T lymphocytes was evidenced, with an inversion of the CD4/CD8 ratio (CD4 14.9% vs 15.5% p=0.9 and CD8 49.7% vs 54.5%p=0.5 in ATG and PTCY respectively). We identified a CD4+/CD8+ double-positive T cell (DP) population, which was higher in PTCY group compared to ATG on days +30 (3.5% vs 10.2%, p=0.009), +90 (3.1% vs 12% p= 0.01) and +180 (5.2% vs 13%, p=0.007, in ATG and PTCY respectively ). On day +30 there were higher proportion of T cells (19.9% vs 9.6% p=0.004), B cells (0.76% vs 0.2%, p=0.006) and NK cells (19.2% vs 8.6% p=0.006) in ATG group compared to PTCY and monocytes were higher in PTCY (31.7 vs 44.5%, p=0.02). On day +180 the proportion of B cell were higher in PTCY (6.1% vs 16.6% p=0.014) and the NK cells were higher in the ATG (22.4% vs 14.7% p=0.021) (Figure 1), with higher CD56dim population in ATG group (46% vs 33.6% p=0.0079), while in PTCY there were a higher NK population CD56high on day +60 (40.3% vs 61%, p=0.014). We identified a peripheral expansion of a CD56negCD16posNKG2Dpos NK cell population, which was higher in the PTCY group on day +180 (3.7% vs 13.2% p=0.017). Regarding CMV-specific immune response, we found no difference between the groups on days +30 (5 vs 5 spots/10 4 PBMC p=0.41), +60 (10 vs 6 spots/10 4 PBMC p=0.38), +90 (13 vs 4 spots/10 4PBMC p=0.33) or +180 (7 vs 6 spots/10 4 PBMC p=0.57). Conclusions: In the context of unrelated HSCT, we found no difference in CMV-specific immune response with the use of PTCY or ATG protocol. Post-transplant immunosuppression with PTCY compared to ATG protocol is related to lower levels of T cells, NK cells and B cells on day +30 and later neutrophil engraftment, but with a higher proportion of double positive T cells on days +30, +90 and +180 and a higher proportion of B cells on day +180. At 6 months after transplantation, a proportionately lower number of NK cells, but higher proportion of CD56negCd16posNKG2D NK population was observed in the PTCY group.

424.4: The Protection From CMV Infection in Solid Organ Transplants Is Highly Dependent on CMV T-Cell Specific Immunity And Type of Organ Transplant

Introduction: The CMV specific immune recovery is known to control viral infection and disease. The CMV specific cell-mediated immunity (CMI) and CMV specific humoral immune response (IgG titer and IgG avidity) was explored as candidate biomarker predictive of CMV infection. Methods: 653 observations from 297 SOT including 103 kidney (K), 60 liver (L), 47 heart (H), 87 Lung (Lu) transplants. The follow-up was 0-400 days after transplant. The following parameters were examined: 1) CMV serostatus before transplant 2) CMV IgG titer 3) CMV IgG avidity 4) CMV-ELISPOT 5) primary (D+/R-) or non-primary (D±/R+) CMV infection 6) CMV viremia. Data were statistically analyzed using ANOVA and linear regression analysis and spline model. Results: The main findings of the study are: 1) CMV IgG titers and avidity are not predictive biomarker for CMV infection. The CMV IgG titer and IgG avidity levels are comparable either in infected and non-infected patients. 2) CMV viral load is statistically different between infected D±/R+ and D+/R. The viral load in infected D+/R- is 1 Log higher compared to D±/R+ (10^5 vs 10^4 respectively) 4) The CMV immune reconstitution is more rapid and efficient in D±/R+ compared to D+/R- 5) D±/R+ and D+/R- Kidney and Lung transplants display a statistically significant lower CMI compared to heart and liver transplants; 6) Kidney and Lung transplants display a similar pattern of immune recovery 7) The CMV cell mediated immunity biomarker is predictive of infection in all organs with exception of lung transplants. Conclusions: The study shows a marginal role of humoral immunity in controlling CMV infection. The pattern of CMI immune recovery is highly dependent upon pre-transplant CMV serostatus, type of organ transplant and immunosuppression therapy. High levels of CMV specific cell-mediated immunity correlate with a reduced CMV infection risk in heart, kidney and liver transplants. A different scenario emerged in lung transplants: high levels of circulating CMV specific T-cells can be found in lung transplants but cannot prevent CMV infection and clear the virus. In this view the lung may be considered a “sanctuary site” for CMV replication.

Humoral/Cellular Immune Discordance in Stem Cell Donors: Impact on Cytomegalovirus-Specific Immune Reconstitution after Related Hematopoietic Transplantation

Cytomegalovirus (CMV) reactivation is an important cause of complications after hematopoietic stem cell transplantation (HSCT). Discrepancies between serologic and cellular CMV-specific immune response have been reported. This study evaluated the impact of lack of CMV-specific CD8+ T cell response in seropositive donors (ie, discordant donors) on the reconstitution of CMV-specific cell-mediated immunity (CMI) after related HSCT in seropositive recipients. CMV-CMI was assessed in donors and recipients using the QuantiFERON-CMV assay (QF). CMV-CMI was prospectively assessed for 1 year in 81 CMV-seropositive HSCT recipients with a haploidentical or matched related donor. A Cox proportional hazard regression analysis was performed. Of the 67 CMV-seropositive donors, 54 (80.6%) were D+QFpos. The remaining 13 CMV-seropositive donors (19.4%) had a QFneg result and thus were classified as discordant donors (D+QFneg). We found that patients with D+QFneg had a significantly higher risk of impaired CMV-CMI reconstitution compared with patients with D+QFpos (log-rank test, P=.001) or D- donors (log-rank test, P = .023). In addition, the D+QFneg group had a higher incidence of single-episode reactivation compared with D+QFpos or D- donors (69.2% versus 44.4% and 28.6%, respectively) but a lower incidence of CMV recurrence compared with the D- group (7.7% versus 57.1%; P=.003). After adjusting for other relevant variables, immune discordance in donors was independently associated with impaired CMV-CMI reconstitution compared with D+QFpos donors (adjusted hazard ratio [HR], 0.18; 95% confidence interval [CI], .06 to .52; P=.001) and D- donors (adjusted HR, .17; 95% CI, .05 to .59; P=.005). Discordant donors were associated with undetectable CMV-CMI during the 12-month follow-up period using the QF assay. The inability of these patients to become QFpos persisted even after CMV reactivation. This might be related to the low frequency of CMV recurrence in this group. CMV-CMI assessment, in conjunction with CMV serostatus, can be of utility to better classify stem cell donors as well as the risk of impaired CMV-CMI reconstitution after HSCT.

CMV Triplex Vaccine to Enhance Adaptive NK and T-cell Reconstitution After Autologous Hematopoietic Cell Transplantation

Cytomegalovirus (CMV) reactivation after hematopoietic cell transplantation (HCT) augments adaptive (CD56dimNKG2C+CD57+) natural killer (NK) and CMV-specific T cells, with potential antitumor effects. Our recent work found an association between higher abundance of adaptive NK cells after auto-HCT and lower risk of relapse in patients with multiple myeloma. Triplex vaccine is a recombinant modified vaccinia Ankara expressing immunodominant CMV antigens, which significantly enhanced CMV-specific T-cell immune responses in allo-HCT recipients. We evaluated whether 2 doses of the vaccine after auto-HCT in patients with lymphoma or myeloma improves reconstitution of adaptive NK and CMV-specific T cells. The primary endpoint was the number of adaptive NK cells at day 100 (∼1 month after dose 2) relative to day 28 (before dose 1). We conducted a single-arm phase 2 clinical trial of 20 patients with lymphoma or myeloma undergoing auto-HCT. Two doses of the vaccine were given on days 28 and 56. Adaptive NK cells increased in CMV-seronegative patients (P=.02), a rise that was more substantial than in unvaccinated historical CMV-seronegative cohorts (P=.03 comparing the rise between the 2 cohorts). There was also an increase in both CD4+ and CD8+ CMV-specific T cells in CMV-seronegative patients (P=.01) and CMV-specific CD8+ effector T cells in CMV-seropositive patients (P=.03). Triplex vaccine improved reconstitution of adaptive NK and CMV-specific T cells after auto-HCT in patients with lymphoma and myeloma. Further study is needed to determine the clinical impact of this modulation of immune response.

CytomegaloVirusDb: Multi-omics knowledge database for cytomegaloviruses

Cytomegalovirus infection is a significant health concern and need further exploration in immunologic response mechanisms during primary and reactivated CMV infection. In this work, we evaluated the whole genomes and proteomes of different CMV species and developed an integrated open-access platform, CytomegaloVirusDb, a multi-Omics knowledge database for Cytomegaloviruses. The resource is categorized into the main sections “Genomics,” “Proteomics,” “Immune response,” and “Therapeutics,“. The database is annotated with the list of all CMV species included in the study, and available information is freely accessible at http://www.cmvdb.dqweilab-sjtu.com/index.php. Various parameters used in the analysis for each section were primarily based on the whole genome or proteome of each specie. The platform provided datasets are open to access for researchers to obtain CMV species-specific information. This will help further to explore the dynamics of CMV-specific immune response and therapeutics. This platform is a useful resource to aid in advancing research against Cytomegaloviruses.

Low frequency of cytomegalovirus (CMV) disease despite high prevalence of CMV viraemia in patients with advanced HIV infection: a clinical and immunological 48-week follow-up study.

The aim of the study was to investigate the dynamics of cytomegalovirus (CMV) replication and CMV-specific immune response recovery after antiretroviral treatment (ART) initiation in patients with advanced HIV infection. A prospective observational study of patients with HIV infection and CD4 counts of <100 cells/µL was carried out (September 2015 to July 2018). HIV viral load (VL), CD4 count and CMV VL were determined by quantitative polymerase chain reaction (PCR) at baseline and at 4, 12, 24 and 48weeks, and CMV-specific immune response was determined by QuantiFERON-CMV assay at baseline and 48weeks. All patients were started on ART but only those with CMV end-organ disease (EOD) received anti-CMV treatment. Fifty-three patients with a median age of 43.6 [interquartile range (IQR) 36.7-52.4] years were included in the study. At baseline, the median CD4 count was 30 cells/µL (IQR 20-60 cells/µL) and the median HIV VL was 462000 HIV-1 RNA copies/mL (IQR 186000-1300000 copies/mL). At baseline, 32% patients had detectable CMV viraemia but none had detectable CMV viraemia at 48weeks. Only one of 53 (1.9%) patients developed EOD during follow-up. Seven (13.2%) patients were lost to follow-up and six (11.3%) died; none of the deaths was related to CMV. Similar percentages of patients had a CMV-specific immune response at baseline (71.7%) and at 48 weeks (70.0%). The magnitude of this response tended to increase over time [median 1.63 (IQR 0.15-5.77) IU/mL at baseline vs. median 2.5 (IQR 0.1-8.325) IU/mL at 48weeks; P=0.11]. We did not find any risk factors associated with 48-week mortality. Although the prevalence of CMV viraemia in patients with advanced HIV infection remains high, achieving a good immunological recovery through ART is enough to suppress CMV viraemia, without an increased risk of CMV EOD. The prevalence of a CMV-specific immune response was high and endured over time.

Is It Feasible to Use CMV-Specific T-Cell Adoptive Transfer as Treatment Against Infection in SOT Recipients?

During the last decade, many studies have demonstrated the role of CMV specific T-cell immune response on controlling CMV replication and dissemination. In fact, it is well established that transplanted patients lacking CMV-specific T-cell immunity have an increased occurrence of CMV replication episodes and CMV-related complications. In this context, the use of adoptive transfer of CMV-specific T-cells has been widely investigated and applied to Hematopoietic Stem Cell Transplant patients and may be useful as a therapeutic alternative, to reconstitute the CMV specific T-cell response and to control CMV viremia in patients receiving a transplantation. However, only few authors have explored the use of T-cell adoptive transfer in SOT recipients. We propose a novel review in which we provide an overview of the impact of using CMV-specific T-cell adoptive transfer on the control of CMV infection in SOT recipients, the different approaches to stimulate, isolate and expand CMV-specific T-cells developed over the years and a discussion of the possible use of CMV adoptive cellular therapy in this SOT population. Given the timeliness and importance of this topic, we believe that such an analysis will provide important insights into CMV infection and its treatment/prevention.

1183. A Prospective Epidemiological &amp; Immunological Study of Cytomegalovirus infection in SLE Patients Receiving Immunosuppressant

BackgroundCytomegalovirus (CMV) infection has been emerging among autoimmune disease patients. CMV-specific cellular immunity has been shown to be correlated with CMV clearance in transplant recipients, however, this correlation has not been assessed in autoimmune disease patients. We aimed to investigate the epidemiology and clinical variables of CMV infection focused on CMV-specific cellular response of CMV infection in active systemic lupus erythematosus (SLE) patients receiving immunosuppressant.MethodsA prospective cohort study of active SLE patients who received immunosuppressant and underwent preemptively monitored for CMV infection by quantitative real-time PCR (Abbott RealTime CMV assay). Clinical variables were collected. CMV-specific cellular immune responses were measured by an enzyme-linked immunosorbent assay (QuantiFERON-CMV®) and intracellular cytokine assay. Clinically significant CMV infection was defined as CMV disease or plasma CMV DNA loads > 1,000 IU/mL.ResultsWe included 45 active SLE patients in our cohort. Among 39 evaluable patients, there were 36 (92%) female patients with a median age of 28 (IQR 24-44) years. A median SLEDAI score was 16 (IQR 9-20). Seventy-four % had renal involvement. Methylprednisolone was the most common immunosuppressive regimen (94.9%). Thirty-six (92.3%) patients were CMV-seropositive. Clinically significant CMV infection occurred in 6 (25.6%) patients included asymptomatic CMV infection (79.3%), CMV syndrome (17.3%), and CMV tissue invasive disease (3.4%). Among 197 blood samplings, there were 16 (8.1%) episodes of clinically significant CMV infection. The distributions of QuantiFERON-CMV® status were reactive, non-reactive and indeterminate (33% vs. 44% vs. 22%; p = 0.56), respectively. The percentage of CMV-IE1-specific NKT cells in those with clinically significant CMV infection were lower than those without infection (0.18% vs. 1.93%, p = 0.03). In multivariate analysis, neurological involvement was associated with clinically significant CMV infection (OR 7.9; 95% CI 1.5-41.9, [p = 0.015]).ConclusionActive SLE patients with neurological involvement who received intense immunosuppression are at risk of CMV infection. Lack of CMV-specific NKT cell response tends to be associated with CMV infection.Disclosures All Authors: No reported disclosures

Vaccination of Hematopoietic Stem Cell (HCT) Donors with Triplex, a Novel Cytomegalovirus Vaccine: Safety, Feasibility and Adoptive Transfer of Protective CMV-Specific T Cells in HCT Recipients

An innovative approach to protect hematopoietic cell transplant (HCT) recipients from cytomegalovirus (CMV) viremia early after transplant is vaccinating the donor prior to stem cell harvest, before the recipient can achieve adequate immune responses. Triplex vaccine, a recombinant modified vaccinia Ankara expressing immunodominant CMV antigens (pp65, IE1 and IE2), is being evaluated in a Phase 1 trial (NCT03560752) to immunize donors of CMV seropositive (CMV+) recipients. Triplex, developed to elicit and enhance protective CMV-specific T cells, is a promising vaccine that demonstrated excellent tolerability and immunogenicity in healthy adults (La Rosa et al., Blood 2017), and in HCT CMV seropositive recipients, who had a 50% reduction in CMV reactivation requiring preemptive antiviral therapy, and significantly enhanced CMV-specific immune responses (Aldoss et al. AIM, 2020). Aim of the current Phase I trial is to assess a proof of concept and preliminary results of safety, feasibility and transfer of CMV-specific immunity, in recipients receiving an HCT from a matched related donor (MRD), vaccinated with Triplex. After obtaining informed consent, 18-75 year old HCT recipients (CMV seropositive) with matched related donors underwent T cell replete HCT. Donors received one Triplex injection 10-60 days prior to start of stem cell mobilization, and recipients underwent HCT within 9 weeks of donor vaccination. As per standard of care, Triplex vaccinated donors were mobilized with granulocyte colony-stimulating factor, prior to apheresis. The study has a target of 18 HCT donor/recipient pairs. All HCT recipients are followed until day 365 post-HCT for safety, virologic and immunologic assessment. All donors are monitored for vaccine induced adverse events. Antiviral treatment for viremia is considered a failure of donor vaccination to provide protection to the recipient. As of July 2020, we have safely vaccinated 9 MRD with Triplex, and infused mobilized peripheral blood stem cells, with T cells by volume approximately 10-25% to their recipients. No adverse events, possibly or likely related to the vaccine were reported. No preemptive antiviral treatment was administered to any of the HCT recipients. Six donor/recipient pairs have reached study end, and immune monitoring has been completed. Two donor/recipient pairs have been evaluated immunologically for up to 6 months and one to day 100 post-HCT. The flow cytometry panel of cellular immune assays included measuring concentrations of CMV-specific T cells expressing the 4-1BB (CD137) activation marker, and assessing the memory phenotype profiles (performed as detailed in La Rosa et al., Blood 2017). Peripheral blood mononuclear cells (PBMC) were stimulated for 24 hours with either pp65, IE1 and IE2 overlapping 15mer peptide libraries and then stained with fluorescently tagged antibodies against CD137, CD3, CD8, CD4, combined with CD28 and CD45RA memory markers by using a Beckman-Coulter Gallios cytometer with Kaluza software. Early post-HCT development of robust and long-lasting frequency of pp65-, IE1- and IE2-specific CD4 and CD8 T cells was observed in all recipients (Figure 1A). Memory profiles of CMV specific T cells had marked prevalence of effector memory phenotype early post-HCT, which persisted over time. Interestingly, on day 28 post-HCT, pp65-specific CD8 T cell levels were significantly higher in the 9 recipients who received an HCT from a Triplex vaccinated MRD compared to a control cohort of consecutively enrolled MRD HCT recipients (Figure 1B). In summary, this is the first clinical trial to assess the possible benefit of vaccinating MRD, to elicit early CMV immune recovery in CMV seropositive HCT recipients, at high risk for developing CMV serious complication after HCT. The approach is feasible and highly tolerable, with promising evidence of efficacy, indicating that Triplex vaccination of the MRD confers early protective anti-CMV immunity to the HCT recipient. Clinical and immunological outcomes from the ongoing study indicate that this novel immune therapeutic strategy provides a safer alternative to antivirals, including letermovir prophylaxis and may find application in higher CMV risk patients, such as haploidentical HCT recipients. Figure 1 Disclosures Nakamura: Viracor: Consultancy; Celgene: Other: Support on seminar; Kyowa-Kirin: Other: Support on a meeting presentation; Alexion: Other: Support on a meeting presentation; Merck: Other: advisory board meeting; Magenta Therapeutics: Other: Advisory board meeting; NapaJen Pharma: Consultancy; Kadmon Corporation: Other: Advisory board meeting. Diamond:Helocyte: Consultancy.

Role of Neutralizing Antibodies in CMV Infection: Implications for New Therapeutic Approaches.

Cytomegalovirus (CMV) infection elicits a potent immune response that includes the stimulation of antibodies with neutralizing activity. Recent studies have focused on elucidating the role of neutralizing antibodies in protecting against CMV infection and disease and characterizing viral antigens against which neutralizing antibodies are directed. Here, we provide a synthesis of recent data regarding the role of neutralizing antibodies in protection against CMV infection/disease. We consider the role of humoral immunity in the context of the global CMV-specific immune response, and the implications that recent findings have for vaccine and antibody-based therapy design.

Lack of cytomegalovirus (CMV)-specific cell-mediated immune response using QuantiFERON-CMV assay in CMV-seropositive healthy volunteers: fact not artifact

The QuantiFERON-CMV (QF) assay measures cell-mediated immunity against cytomegalovirus (CMV-CMI), which is particularly useful in individuals susceptible to CMV infection such as transplant patients. A positive QF result identifies patients that are better protected against CMV infection. However, the significance of a negative QF result in CMV-seropositive individuals needs to be clarified. CMV-CMI was analyzed in healthy subjects using the QF assay, and, in parallel, the Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA). FASCIA assay measures T-cell proliferation using CMV lysate as stimulus whereas QF assay use a mix of peptides. A total of 93 healthy volunteers were enrolled, and 13/71 CMV-seropositive individuals (18.3%) showed humoral/cellular discordance using QF assay (CMV+ QF−). Interestingly, with FASCIA assay CD4+ and CD8+ T-cell proliferations were lower in CMV+ QF− than in CMV+ QF+ individuals. Furthermore, CMV+ QF− volunteers had a lower level of anti-CMV IgG than CMV+ QF+ subjects. Discordant CMV+ QF− volunteers can be defined as low responder individuals since they show lower CMV-specific humoral and cellular immune responses in comparison to CMV+ QF+ individuals. Immune discordance shows the high heterogeneity of immunity to CMV in healthy subjects.

Cytomegalovirus (CMV)-Specific Polyfunctional T-Cell Responses after Letermovir (LET) Prophylaxis in Hematopoietic Cell Transplantation (HCT)

<h3>Introduction</h3> In a randomized, placebo-controlled trial, prophylaxis with LET decreased clinically significant CMV infection after allogeneic HCT; however, there was an increase of CMV infection after discontinuation of prophylaxis at day 100 (NEJM 2017; 377:2433). <h3>Objective</h3> To determine the influence of LET prophylaxis on CMV-specific immune reconstitution after HCT as a possible mechanism for the observed effect, we compared CMV T-cell responses in a prospective cohort of allogeneic HCT recipients who received LET prophylaxis to CMV T-cell responses in historical controls transplanted prior to the introduction of LET. <h3>Methods</h3> We included CMV-seropositive adult patients given LET prophylaxis after first allogeneic HCT; controls received PCR-guided preemptive CMV-directed therapy prior to the introduction of LET (2010-17). PBMC samples collected ∼3 months post-HCT were cryopreserved and then analyzed for intracellular expression of TNF-α, IL-2, IFN-γ, CD107a, and Granzyme B in CD4 and CD8 T cells. Samples stimulated with CMV peptide mixes (pp65 and IE-1) and staphylococcal enterotoxin B (SEB) served as positive controls. Clinical data including baseline characteristics were obtained from medical records. Polyfunctional T-cell responses were defined as those expressing IFN-γ plus at least one additional cytokine. Univariable Wilcoxon ranksum tests were used to compare differences between LET patients and controls and multivariate linear regression analysis was performed to adjust comparisons for other factors affecting immune reconstitution, such as donor CMV serostatus, lymphopenia, and GVHD severity. <h3>Results</h3> Forty-two patients given LET and 95 controls were evaluated; patient characteristics were similar between groups. Polyfunctional CD8 T-cell responses at 3 months were lower in LET patients under all testing conditions while CD4 T-cell responses were decreased after stimulation with IE-1 only (Figures 1-3). After adjusting for CMV donor status, lymphopenia, GVHD and underlying disease, polyfunctional CD8 (Mean difference -5.736, 95% Confidence interval [CI] -11.17, -0.301, p=0.039) and CD4 (Mean difference -0.138, 95% CI -0.257, -0.019, p=0.023) T-cell responses to IE-1 remained significantly decreased in patients given LET prophylaxis compared to controls. <h3>Conclusion</h3> LET prophylaxis appeared to be associated with a delayed CMV-specific cellular immune response. Our study provides a potential mechanism for the observed increase of clinically significant CMV events after discontinuation of LET prophylaxis. Studies are ongoing to evaluate correlations of the observed CMV-specific immunodeficiency with post-day 100 CMV events. Measuring CMV-specific T cell responses at the conclusion of LET prophylaxis could identify patients who might benefit from extended surveillance or prolonged prophylaxis.

Cytomegalovirus-specific CD8+ T-cell responses are associated with arterial blood pressure in people living with HIV.

People living with HIV (PLHIV) are at increased risk for cardiovascular disease (CVD), and immunity against cytomegalovirus (CMV) may be a contributing factor. We hypothesized that enhanced T-cell responses against CMV and CMV-IgG antibody-levels are associated with higher arterial blood pressure in PLHIV. We assessed serum CMV-IgG, systolic- (SBP) and diastolic- (DBP) blood pressure, pulse pressure (PP), traditional risk factors, activated CD8+ T-cells (CD38+HLA-DR+), senescent CD8+ T-cells (CD28-CD57+) and interleukin-6 (IL-6) in 60 PLHIV and 31 HIV-uninfected controls matched on age, gender, education and comorbidity. In PLHIV, expression of interleukin-2, tumor necrosis factor-α and interferon-γ was measured by intracellular-cytokine-staining after stimulation of T-cells with CMV-pp65 and CMV-gB. Associations between CMV-specific immune responses and hypertension, SBP, DBP or PP were assessed by multivariate logistic and linear regression models adjusted for appropriate confounders. The median age of PLHIV was 47 years and 90% were male. Prevalence of hypertension in PLHIV was 37% compared to 55% of HIV-uninfected controls. CMV-specific CD8+ T-cell responses were independently associated with higher PP (CMV-pp65; β = 2.29, p = 0.001, CMV-gB; β = 2.42, p = 0.001) in PLHIV. No significant differences were found with regard to individual measures of SBP and DBP. A possible weak association was found between CMV-IgG and hypertension (β = 1.33, p = 0.049) after adjustment for age, smoking and LDL-cholesterol. HIV-related factors, IL-6, CD8+ T-cell activation or CD8+ T-cell senescence did not mediate the associations, and no associations were found between CMV-specific CD4+ T-cell responses and blood pressure in PLHIV. In conclusion, increased arterial blood pressure in PLHIV may be affected by heightened CMV-specific CD8+ T-cell responses.

Clinical Implications of the CMV-Specific T-Cell Response and Local or Systemic CMV Viral Replication in Patients With Moderate to Severe Ulcerative Colitis.

BackgroundThe precise role of cytomegalovirus (CMV) in ulcerative colitis (UC) remains disputed. We evaluated the association of CMV-specific host immune responses and systemic or local viral replication with responses to systemic steroids in patients with moderate to severe UC.MethodsPatients who were hospitalized for moderate to severe UC between April 2015 and June 2016 were enrolled. At baseline, all enrolled patients underwent CMV-specific enzyme-linked immunospot assays, quantitative polymerase chain reaction (qPCR) analysis of blood and colonic tissue for CMV viral load, histopathological testing for CMV in colonic tissue by hematoxylin and eosin staining, and immunohistochemical (IHC) analysis. Clinical responses to steroid therapy based on the Oxford index were assessed on day 3.ResultsOf the 80 patients evaluated, 28 (35.0%) had poor responses to steroid therapy on day 3 of intensive treatment. The presence of inclusion bodies (32.1%) and high-grade (≥3) positivity on IHC (50.0%), as well as colonic (mean 1440.4 copies/mg) and blood (mean, 3692.6 copies/mL) CMV viral load, were higher in steroid-refractory UC patients than the control group (13.5%, 1.9%, mean 429.2 copies/mg, and mean 231.2 copies/mL, respectively; P = .046, .009, .017, and .002, respectively). However, CMV-specific T-cell responses were not associated with steroid-refractory UC. Multivariate analysis revealed that a higher Mayo score (odds ratio [OR], 2.00; P = .002) and higher blood CMV viral load via qPCR analysis (OR, 3.58; P = .044) were independent risk factors for steroid-refractory UC.ConclusionsIn patients with moderate to severe UC, higher Mayo score and blood CMV expression determined by qPCR are independently associated with steroid refractoriness.ClinicalTrials.gov registration numberNCT 02439372.

Quantifying Cytomegalovirus-Specific T Cell Responses Prior to Hospital Discharge Allows Risk Stratification for Late Viral Reactivation after Allogenic Hematopoietic Cell Transplantation

Background Cytomegalovirus (CMV) infection remains a significant complication after allogenic hematopoietic cell transplantation (HCT). In the first three months after HCT, close monitoring of patients associated with prophylactic or preemptive administration of ganciclovir effectively prevents CMV-related complications. However, once patients are discharged from cancer centers around day 100 after HCT their risk of late CMV reactivation remains high. Since virus-specific T cells are capable of controlling CMV replication, we assessed whether their quantification shortly before hospital discharge allows risk-stratification for late CMV reactivation. Methods Allogeneic HCT recipients of all ages at the Fred Hutchinson Cancer Research Center who were CMV-seropositive or had a CMV-seropositive donor and participated in a prospective immune monitoring study were analyzed if they received at least 50% of weekly scheduled viral load (VL) PCR tests between days 100 and 180 after transplantation. CMV-specific cellular immunity was quantified via interferon-gamma ELISpot assay from cryopreserved peripheral blood mononuclear cells (PBMC) that were collected around day 90 after HCT. Cells were stimulated with peptide pools spanning the entire IE-1 and pp65 proteins (15mers with 11 amino acid overlap). Univariate Cox regression analysis was performed to determine hazard ratios (HR) for developing CMV reactivation at ≥50 IU, ≥500, ≥1000 IU/mL using different threshold levels (10, 50, or 100 spot-forming units (SFU) per 200,000 PBMC) for IE-1 and pp65. Results 13 out of 69 patients (18.8%) in this cohort showed clinically significant viral reactivation between days 100 and 180 after HCT, defined as having a viral load ≥1000 IU/ml at least once during the observational period (this level is commonly used to start preemptive treatment in the late setting). The best separation of risk was achieved by using a threshold of 10 SFU for both antigens: Individuals with less than 10 SFU for IE-1 or pp65 had an increased risk for CMV reactivation (HR 8.49, 95% CI, p=0.04) compared to patients with at least 10 SFU for both antigens. Correspondingly, the cumulative incidence of CMV reactivation was significantly higher in the group of patients with Conclusions Quantification of CMV-specific cellular immune responses around day 100 can help identify patients that are at risk for developing clinically significant late CMV reactivation. Both IE-1 and pp65 are useful for predicting CMV reactivation. Further studies with larger cohorts should be performed to confirm the suggested threshold levels of immune protection.

CMV-Specific Immune Response-New Patients, New Insight: Central Role of Specific IgG during Infancy and Long-Lasting Immune Deficiency after Allogenic Stem Cell Transplantation.

Although the existing paradigm states that cytomegalovirus (CMV) reactivation is under the control of the cellular immune response, the role of humoral and innate counterparts are underestimated. The study analyzed the host–virus interaction i.e., CMV-immune response evolution during infection in three different clinical situations: (1) immunodeficient CMV-positive human leukocyte antigen (HLA)-matched bone marrow recipients after immunoablative conditioning as well as immunocompetent, (2) adult, and (3) infant with primary immune response. In the first situation, a fast and significant decrease of specific immunity was observed but reconstitution of marrow-derived B and natural killer (NK) cells was observed prior to thymic origin of T cells. The lowest CMV-IgG (93.2 RU/mL) was found just before CMV viremia. It is noteworthy that the sole and exclusive factor of CMV-specific immune response is a residual recipient antibody class IgG. The CMV-quantiferon increase was detected later, but in the first phase, phytohemagglutinin (PHA)-induced IFN-γ release was significantly lower than that of CMV-induced (“indeterminate” results). It corresponds with the increase of NK cells at the top of lymphocyte reconstitution and undetected CMV-specific CD8 cells using a pentamer technique. In immunocompetent adult (CMV-negative donor), the cellular and humoral immune response increased in a parallel manner, but symptoms of CMV mononucleosis persisted until the increase of specific IgG. During infancy, the decrease of the maternal CMV-IgG level to 89.08 RU/mL followed by clinical sequel, i.e., CMV replication, were described. My observations shed light on a unique host-CMV interaction and CMV-IgG role: they indicate that its significant decrease predicts CMV replication. Before primary cellular immune response development, the high level of residual CMV-IgG (about >100 R/mL) from mother or recipient prevents virus reactivation. The innate immune response and NK-dependent IFN-secretion should be further investigated.

638. CMV-Specific T-Cell Immune Responses in Older vs. Younger Kidney Transplant Recipients

BackgroundCompared with younger patients on similar immunosuppression regimens, older solid-organ transplant recipients experience increased rates of infection and death, but decreased rates of rejection. The mechanism behind these differences has yet to be defined, but may be related to ÒinflammagingÓ driven by CMV infection. The objective of this study was to evaluate older vs. younger solid-organ transplant recipients for CMV-specific T-cell immune responses.MethodsPeripheral blood mononuclear cells were isolated from 20 older (3 age 60) and 25 matched younger (ages 30–59) kidney transplant recipients at 3 months after transplantation. Eight recipients were high risk by CMV serology (D+/R−) and 37 were intermediate risk (D−/R+). Overlapping CMV peptide pools were used for stimulation. Intracellular staining to determine cytokine stimulation was performed by multiparameter flow cytometry. Statistical analysis was performed using Jmp Pro 11 software.ResultsThere was no association between patient age and CMV risk status (P = 0.728). There was no difference between older and younger kidney transplant recipients in release of IFNγ, TNFα, or IL-2 from CD4+ or CD8+ T cells in response to CMV antigen stimulation. However, Older recipients had similar frequencies of CD8+ naive cells but decreased frequency of CD8+ terminally differentiated effector memory CD45RA+ (TEMRA) T cells releasing both IFNg and TNFa (P = 0.037) (figure). Interestingly, development of CMV viremia was associated with a weaker CMV-specific immune response: Patients who had a history of CMV viremia had a decreased frequency of CD8+ TEMRA cells releasing both IFNγ and TNFα (P = 0.041).ConclusionOlder kidney transplant recipients demonstrated a decreased frequency of CMV-specific polyfunctional CD8+ TEMRA T cells. This impaired memory T-cell response to CMV suggests a possible mechanism for the increased vulnerability of older recipients to CMV infection or reactivation, which may in turn worsen age-related immune dysfunction. Furthermore, patients with subsequent CMV viremia had a decreased frequency of CMV-specific polyfunctional CD8+ TEMRA T cells. This finding may explain patient vulnerability to CMV viremia despite modern protocols for antiviral prophylaxis. Disclosures All authors: No reported disclosures.