Reactivation Of Latent Cytomegalovirus Research Articles

Patient Outcomes in CMV and SARS-CoV-2 Coinfection

Abstract This study examines clinical outcomes in patients with cytomegalovirus (CMV) and SARS-CoV-2 coinfection. Between June and November 2020, previously immunocompetent patients with SARS-CoV-2 and CMV coinfection were identified at Houston Methodist Hospital as part of routine clinical correlation by a molecular pathologist. SARS-CoV-2 nasopharyngeal specimens were analyzed by real time reverse-transcriptase polymerase chain reaction (RT-PCR). All CMV tests were performed on plasma or bronchoalveolar lavage (BAL) specimens and analyzed by competitive polymerase chain reaction. 65 previously immunocompetent patients with CMV and SARS-CoV-2 coinfection were identified. Patient demographics include 41 male patients (63%) and 24 female patients (37%) ranging in age from 34 to 86 years (mean: 66.04, median 68). Documented pre-existing conditions include 27 patients with hypertension 41.5%), 19 patients with diabetes mellitus (29.2%), 9 patients with coronary artery disease (13.8%), and 3 patients with asthma (4.6%). Eight patients (12.3%) had no documented pre-existing conditions. The plasma CMV viral load ranged from <300 to 21,566 IU/mL. The CMV PCR results from bronchoalveolar lavage and bronchial wash specimens ranged from <300 to 59,127 IU/mL. CMV PCR was initially negative in 10 patients then positive on serial testing. 60 patients were critically ill requiring ventilator support (92.3%). 47 patients (72.3%) expired, 7 patients (10.8%) were transferred to a long term acute care facility, 3 patients (4.6%) were discharged to a rehabilitation facility, 3 patients (4.6%) were discharged home, and 1 patient (1.5%) remained in-patient at the time of analysis. The prevalence of CMV seropositivity and medical comorbidities increases with age. Reactivation of latent CMV is a known occurrence in critically ill patients that is associated with poor outcomes. The majority of the patients in our cohort were 50 years old, and all were severely to critically ill with a mortality rate of 72.3% These findings suggest CMV portends a worse prognosis in patients with COVID-19. These findings also demonstrate the importance of clinical correlation in molecular testing.

Therapeutic Vaccination of Hematopoietic Cell Transplantation Recipients Improves Protective CD8 T-Cell Immunotherapy of Cytomegalovirus Infection.

Reactivation of latent cytomegalovirus (CMV) endangers the therapeutic success of hematopoietic cell transplantation (HCT) in tumor patients due to cytopathogenic virus spread that leads to organ manifestations of CMV disease, to interstitial pneumonia in particular. In cases of virus variants that are refractory to standard antiviral pharmacotherapy, immunotherapy by adoptive cell transfer (ACT) of virus-specific CD8+ T cells is the last resort to bridge the “protection gap” between hematoablative conditioning for HCT and endogenous reconstitution of antiviral immunity. We have used the well-established mouse model of CD8+ T-cell immunotherapy by ACT in a setting of experimental HCT and murine CMV (mCMV) infection to pursue the concept of improving the efficacy of ACT by therapeutic vaccination (TherVac) post-HCT. TherVac aims at restimulation and expansion of limited numbers of transferred antiviral CD8+ T cells within the recipient. Syngeneic HCT was performed with C57BL/6 mice as donors and recipients. Recipients were infected with recombinant mCMV (mCMV-SIINFEKL) that expresses antigenic peptide SIINFEKL presented to CD8+ T cells by the MHC class-I molecule Kb. ACT was performed with transgenic OT-I CD8+ T cells expressing a T-cell receptor specific for SIINFEKL-Kb. Recombinant human CMV dense bodies (DB-SIINFEKL), engineered to contain SIINFEKL within tegument protein pUL83/pp65, served for vaccination. DBs were chosen as they represent non-infectious, enveloped, and thus fusion-competent subviral particles capable of activating dendritic cells and delivering antigens directly into the cytosol for processing and presentation in the MHC class-I pathway. One set of our experiments documents the power of vaccination with DBs in protecting the immunocompetent host against a challenge infection. A further set of experiments revealed a significant improvement of antiviral control in HCT recipients by combining ACT with TherVac. In both settings, the benefit from vaccination with DBs proved to be strictly epitope-specific. The capacity to protect was lost when DBs included the peptide sequence SIINFEKA lacking immunogenicity and antigenicity due to C-terminal residue point mutation L8A, which prevents efficient proteasomal peptide processing and binding to Kb. Our preclinical research data thus provide an argument for using pre-emptive TherVac to enhance antiviral protection by ACT in HCT recipients with diagnosed CMV reactivation.

Cytomegalovirus Encephalopathy during Brain Tumor Irradiation.

Some patients with brain cancer show extremely short survival postradiochemotherapy treatment for unknown reasons. Recent work shows that this is closely linked to encephalopathy associated with reactivation of latent cytomegalovirus in the host. Importantly, survival can be enhanced by treatment with antiviral drugs.See related article by Goerig et al., p. 3259.

Cytomegalovirus-Associated Inhibition of Hematopoiesis Is Preventable by Cytoimmunotherapy With Antiviral CD8 T Cells.

Reactivation of latent cytomegalovirus (CMV) in recipients of hematopoietic cell transplantation (HCT) not only results in severe organ manifestations, but can also cause “graft failure” resulting in bone marrow (BM) aplasia. This inhibition of hematopoietic stem and progenitor cell engraftment is a manifestation of CMV infection that is long known in clinical hematology as “myelosuppression.” Previous studies in a murine model of sex-chromosome mismatched but otherwise syngeneic HCT and infection with murine CMV have shown that transplanted hematopoietic cells (HC) initially home to the BM stroma of recipients but then fail to further divide and differentiate. Data from this model were in line with the hypothesis that infection of stromal cells, which constitute “hematopoietic niches” where hematopoiesis takes place, causes a local deficiency in essential hematopoietins. Based on this understanding, one must postulate that preventing infection of stromal cells should restore the stroma's capacity to support hematopoiesis. Adoptively-transferred antiviral CD8+ T cells prevent lethal CMV disease by controlling viral spread and histopathology in vital organs, such as liver and lungs. It remained to be tested, however, if they can also prevent infection of the BM stroma and thus allow for successful HC engraftment. Here we demonstrate that antiviral CD8+ T cells control stromal infection. By tracking male donor-derived sry+ HC in the BM of infected female sry− recipients, we show the CD8+ T cells allow for successful donor HC engraftment and thereby prevent CMV-associated BM aplasia. These data provide a further argument for cytoimmunotherapy of CMV infection after HCT.

Insufficient Antigen Presentation Due to Viral Immune Evasion Explains Lethal Cytomegalovirus Organ Disease After Allogeneic Hematopoietic Cell Transplantation.

Reactivation of latent cytomegalovirus (CMV) poses a clinical problem in transiently immunocompromised recipients of hematopoietic cell (HC) transplantation (HCT) by viral histopathology that results in multiple organ manifestations. Compared to autologous HCT and to syngeneic HCT performed with identical twins as HC donor and recipient, lethal outcome of CMV infection is more frequent in allogeneic HCT with MHC/HLA or minor histocompatibility loci mismatch between donor and recipient. It is an open question if a graft-vs.-host (GvH) reaction exacerbates CMV disease, or if CMV exacerbates GvH disease (GvHD), or if interference is mutual. Here we have used a mouse model of experimental HCT and murine CMV (mCMV) infection with an MHC class-I mismatch by gene deletion, so that either HCT donor or recipient lack a single MHC class-I molecule, specifically H-2 Ld. This particular immunogenetic disparity has the additional advantage that it allows to experimentally separate GvH reaction of donor-derived T cells against recipient's tissues from host-vs.-graft (HvG) reaction of residual recipient-derived T cells against the transplanted HC and their progeny. While in HvG-HCT with Ld-plus donors and Ld-minus recipients almost all infected recipients were found to control the infection and survived, almost all infected recipients died of uncontrolled virus replication and consequent multiple-organ viral histopathology in case of GvH-HCT with Ld-minus donors and Ld-plus recipients. Unexpectedly, although anti-Ld-reactive CD8+ T cells were detected, mortality was not found to be associated with GvHD histopathology. By comparing HvG-HCT and GvH-HCT, investigation into the mechanism revealed an inefficient reconstitution of antiviral high-avidity CD8+ T cells, associated with lack of formation of protective nodular inflammatory foci (NIF) in host tissue, selectively in GvH-HCT. Most notably, mice infected with an immune evasion gene deletion mutant of mCMV survived under otherwise identical GvH-HCT conditions. Survival was associated with enhanced antigen presentation and formation of protective NIF by antiviral CD8+ T cells that control the infection and prevent viral histopathology. This is an impressive example of lethal viral disease in HCT recipients based on a failure of the immune control of CMV infection due to viral immune evasion in concert with an MHC class-I mismatch.

Cytomegalovirus antigenemia in patients with autoimmune and non-autoimmune diseases in Beijing: A 10-year single hospital experience.

BackgroundPrimary cytomegalovirus (CMV) infection is prevalent worldwide and usually results in latency in immunocompetent populations. Reactivation of latent CMV can cause life-threatening complications in immunocompromised hosts.MethodsWe used the CMV Brite assay to test CMV antigenemia (pp65) in whole blood samples from 22,192 patients with or without autoimmune diseases in Beijing during 2008–2018.ResultsThe overall prevalence of CMV antigenemia was 19.5% (9.7%, males; 26.0%, females). The prevalence of CMV antigenemia was 35.1%, 58.6% and 11.4% in whole patients with autoimmune diseases, in patients with systemic lupus erythematosus (SLE) and in patients with non-SLE autoimmune diseases, respectively. All patients with non-autoimmune diseases, patients with HIV/AIDS or transplantation were found to have 5.0%, 27% or 14.8%, respectively. Patients≤20 years with SLE had a significantly higher prevalence of CMV antigenemia than did all SLE patients, on average. Patients>51 years with non-SLE autoimmune diseases had a significantly higher prevalence than did all patients with non-SLE autoimmune diseases, on average. The prevalence of CMV antigenemia in patients admitted to intensive-care units (ICUs) were 9.2%, which was significantly higher than that among all patients with non-autoimmune diseases. Patients with SLE had 23.8% of negative conversion of CMV antigenemia, significantly lower than the percentage of patients with non-SLE autoimmune (64.3%) and non-autoimmune (61.0%) diseases. The mean number of days to negative conversion of CMV antigenemia in patients with SLE was 35.3±35.8 days, which was significantly longer than that in patients with non-SLE autoimmune diseases (15.4±11.9 days) and non-autoimmune diseases (13.6±7.7 days).ConclusionsCMV antigenemia is found more likely in women than in men, more prevalently in patients with SLE than those with HIV/AIDS or transplant recipients, more frequently in patients admitted to ICUs. Patients with SLE had prolonged CMV antigenemia. The role of CMV appears important in SLE.

Bullous pemphigoid complicated by cytomegalovirus disease as a manifestation of immune reconstitution inflammatory syndrome: retrospective analyses of our institutional cases and literature review

Cytomegalovirus (CMV) disease induced by reactivation of latent CMV is a fatal viral infection that may develop in a setting of therapy with immunosuppressive agents. There is a clear need to clarify any clinical features and markers of CMV disease. We investigated which clinical markers usually available in a clinical setting can predict CMV disease occurring in bullous pemphigoid (BP) patients receiving corticosteroids. We described a BP patient with CMV disease complicated by gastrointestinal hemorrhage and liver dysfunction. Prompted by this patient, we retrospectively analyzed clinical features and laboratory findings in our institutional four BP patients and previously reported nine BP patients with CMV disease. We also compared these patients with our institutional 42 BP patients not complicated by CMV disease. High levels of anti-BP180 antibody titers associated with resistance to corticosteroids are a risk factor for the development of CMV disease. A reduction in platelet (PLT) and white blood cell (WBC) counts and an increase in alanine aminotransferase (ALT) levels 3-4weeks after the initiation of corticosteroids are useful predictive markers for the onset of CMV disease. Frequent WBC, PLT, and ALT measurements may identify BP patients at a risk of subsequently developing CMV disease. Careful monitoring of CMV disease in BP refractory to systemic corticosteroids may reduce the risk of fatal outcomes.

Dasatinib-Induced CMV Hepatitis in an Immunocompetent Patient: A Rare Complication of a Common Drug

Dasatinib is a common anticancer drug used in the treatment of leukemia. Several side effects have been reported, the most common being myelosuppression, diarrhea, edema, and nausea. Three papers have been published reporting hepatic side effects of dasatinib treatment. A rare side effect of dasatinib treatment is reactivation of latent cytomegalovirus (CMV) infection. Never before has dasatinib therapy shown to be the cause of CMV hepatitis in an immunocompetent patient. We present a case of an immunocompetent patient who was treated with the standard dose of dasatinib therapy and subsequently developed CMV hepatitis. Well-known side effects of dasatinib therapy are understood and documented; unknown adverse drug reactions can occur and should be monitored for. This is a significant finding given the high rate of CMV seropositivity in the general population.

Mast cells: innate attractors recruiting protective CD8 T cells to sites of cytomegalovirus infection.

Reactivation of latent cytomegalovirus (CMV) in the transient immunocompromised state after hematoablative treatment is a major concern in patients undergoing hematopoietic cell transplantation (HCT) as a therapy of hematopoietic malignancies. Timely reconstitution of antiviral CD8 T cells and their efficient recruitment to the lungs is crucial for preventing interstitial pneumonia, the most severe disease manifestation of CMV in HCT recipients. Here, we review recent work in a murine model, implicating mast cells (MC) in the control of pulmonary infection. Murine CMV (mCMV) productively infects MC in vivo and triggers their degranulation, resulting in the release of the CC chemokine ligand 5 (CCL5) that attracts CD8 T cells to infiltrate infected tissues. Comparing infection of MC-sufficient C57BL/6 mice and congenic MC-deficient Kit (W-sh/W-sh) "sash" mutants revealed an inverse relation between the number of lung-infiltrating CD8 T cells and viral burden in the lungs. Specifically, reduced lung infiltration by CD8 T cells in "sash" mutants was associated with an impaired infection control. The causal, though indirect, involvement of MC in antiviral control was confirmed by reversion of the deficiency phenotype in "sash" mutants reconstituted with MC. These recent findings predict that efficient MC reconstitution facilitates the control of CMV infection also in immunocompromised HCT recipients.

Clinical and epidemiological features of congenital cytomegalovirus infection globally

Human cytomegalovirus (CMV) is the most common non-genetic cause of congenital disability. As a herpesvirus that infects the majority of the population, CMV is able to establish a lifelong latent infection in the host. Any time during pregnancy, a primary CMV infection, reactivation of latent CMV or a new viral strain can infect the placenta and the developing foetus, resulting in congenital CMV infection. Each year, an estimated 2000 children are born with congenital CMV infection in Australia, leaving ~500 children with permanent disabilities such as hearing or vision loss, or mental disability. Despite the clinical importance of congenital CMV, there is limited awareness and knowledge in the medical and general community about congenital CMV infection. This article reviews the global epidemiology and clinical features of maternal and congenital CMV infections.

Human Cytomegalovirus Infection in Solid-Organ Transplantation

Human cytomegalovirus (CMV) continues to be a major threat against solid-organ transplant recipients despite significant advancements in its prophylaxis and therapy. Primary CMV infection or reactivation of latent CMV in the transplant recipients may cause CMV diseases such as flu-like viral syndrome and tissue-invasive CMV disease. In addition, CMV infection in the recipients is associated with graft rejection and higher risk of other opportunistic infections, which are collectively known as the indirect effects of CMV infection. Prevention strategies with antiviral drugs including ganciclovir remarkably decreased CMV disease and the indirect effects. Two commonly employed strategies are universal prophylaxis and preemptive therapy. However, gangciclovir-resistant CMV has emerged due to mutations in CMV UL97 and UL54 genes, now requiring alternative therapeutic options to be developed. This review provides an overview of CMV infection and disease, indirect effects on hosts, prevention strategies, and drug resistance in solid-organ transplant recipients.

Antiviral prophylaxis inhibits cytomegalovirus reactivation in critical illness

Reactivation of latent cytomegalovirus (CMV) can lead to viraemia or CMV disease and has been detected in up to 30% of critically ill patients without prior history of immune suppression. However, the clinical importance of this observation remains unclear. We report a proof-of-concept randomised controlled trial of two antiviral drugs in intensive care patients to determine their impact on CMV reactivation.

CMV Reactivation is Associated with Reduced Relapse Risk, Better Disease-Free Survival and Expansion of Adaptive NK Cells after Reduced Intensity Hematopoietic Cell Transplantation

We have previously shown that while NK cells are the first lymphocyte reconstituting after allogeneic transplantation, these NK cells are immature and functionally hyporesponsive when exposed to tumor targets. Remarkably, this can be reversed by reactivation of human cytomegalovirus (CMV). A distinct population of natural killer (NK) cells with attributes of immunological memory arises specifically in response to acute CMV infection in healthy individuals or reactivation of latent CMV in hematopoietic cell transplantation (HCT) recipients. These adaptive NK cells are defined by expression of the activating receptor NKG2C and maturation marker CD57 and display a heightened capacity for inflammatory cytokine production and cytolysis in response to target cell recognition. Since the differentiation and maturation of NK cells is dependent upon interactions with cells of the myeloid lineage, including antigen-presenting cells, we hypothesized that the expansion of adaptive NK cells could influence relapse risk, particularly after a reduced intensity conditioning (RIC) regimen where residual host myeloid cells may intensify donor adaptive NK cell expansion in response to CMV reactivation after HCT. Thus, we analyzed 1-year relapse rates in 674 allogeneic HCT recipients at the University of Minnesota (470 umbilical cord blood (UCB) and 204 matched sibling donor (MSD)) between 2001-2013 with AML (n=313), ALL (n=187), MDS (n=85), CML (n=28), NHL (n=42), Hodgkins (n=14) and Multiple Myeloma (n=5) segregated by recipient CMV serostatus (270 seronegative and 404 seropositive). Within the CMV seropositive group, 190 individuals experienced CMV reactivation. We observed no association between CMV reactivation and 1-year relapse rates in recipients of myeloablative conditioning (n=366; 16% [10%-22%] for CMV seronegative vs. 18% [12-24%] for CMV seropositive without CMV reactivation vs. 15% [8%-22%] for CMV seropositive recipients with CMV reactivation). In marked contrast, CMV reactivation was associated with lower 1-year relapse rates following RIC (n=308; 35% [27%-43%] for CMV seronegative vs. 30% [20-40%] for CMV seropositive without CMV reactivation vs. 26% [17%-35%] for CMV seropositive with CMV reactivation [p for trend=0.05, Figure 1A]). In multivariate analyses adjusting for recipient CMV status, conditioning regimen, donor type (MSD vs. UCB), diagnosis (AML [shown to be sensitive to NK cell effects] vs. other), gender, disease risk (standard vs. high) and prior autologous transplant, CMV reactivation was independently associated with lower relapse risk (RR=0.6 [0.4-1.0], p=0.06) and increased DFS (RR=0.7 [0.5-1.0], p=0.05) following RIC, but not myeloablative HCT. Although CMV reactivation led to superior DFS within the entire cohort (RR=0.7 [0.6-1.0], p=0.04), CMV reactivation was not independently associated with lower relapse risk (RR=1.0 [0.5-1.8], p=0.88) with myeloablative conditioning. To determine whether the preparative regimen also influenced adaptive NK cell differentiation post-transplant, we analyzed the percentage of CD56dimCD57+NKG2C+ NK cells. Compared to myeloablative recipients, after RIC, the percentage of circulating CD56dimCD57+NKG2C+ NK cells was significantly higher at 4 weeks post-CMV reactivation (8.58% vs. 4.64%, p=0.02), 8 weeks post-CMV reactivation (7.77% vs. 3.84%, p=0.02), and 6 months post-transplant (14.80% vs. 6.29%, p=0.01) (Figure 1B). Our results demonstrate a relationship between CMV reactivation and enhanced graft-versus-leukemia (GVL) in patients treated with RIC. The association between lower relapse risk and in vivo expansion and survival of adaptive NK cells suggests that these cells may be critical to reducing the risk of relapse. Strategies to isolate and expand adaptive NK cells in order to enhance the GVL effect without clinical CMV reactivation are being actively explored. [Display omitted] DisclosuresMiller:Coronado: Speakers Bureau; BioSciences: Membership on an entity’s Board of Directors or advisory committees; SAB: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

The histopathological approach to inflammatory bowel disease: a practice guide

Inflammatory bowel diseases (IBDs) are lifelong disorders predominantly present in developed countries. In their pathogenesis, an interaction between genetic and environmental factors is involved. This practice guide, prepared on behalf of the European Society of Pathology and the European Crohn's and Colitis Organisation, intends to provide a thorough basis for the histological evaluation of resection specimens and biopsy samples from patients with ulcerative colitis or Crohn's disease. Histopathologically, these diseases are characterised by the extent and the distribution of mucosal architectural abnormality, the cellularity of the lamina propria and the cell types present, but these features frequently overlap. If a definitive diagnosis is not possible, the term indeterminate colitis is used for resection specimens and the term inflammatory bowel disease unclassified for biopsies. Activity of disease is reflected by neutrophil granulocyte infiltration and epithelial damage. The evolution of the histological features that are useful for diagnosis is time- and disease-activity dependent: early disease and long-standing disease show different microscopic aspects. Likewise, the histopathology of childhood-onset IBD is distinctly different from adult-onset IBD. In the differential diagnosis of severe colitis refractory to immunosuppressive therapy, reactivation of latent cytomegalovirus (CMV) infection should be considered and CMV should be tested for in all patients. Finally, patients with longstanding IBD have an increased risk for the development of adenocarcinoma. Dysplasia is the universally used marker of an increased cancer risk, but inter-observer agreement is poor for the categories low-grade dysplasia and indefinite for dysplasia. A diagnosis of dysplasia should not be made by a single pathologist but needs to be confirmed by a pathologist with expertise in gastrointestinal pathology.

In Vitro Treatment with Ganciclovir Restores the Functionality of Exhausted T Cells from Cancer Patients

Summary Background Human cytomegalovirus (CMV) is a lifelong, often asymptomatic infection, with the virus maintained in latently infected myeloid cells. Patients receiving chemotherapy for cancer are at high risk of reactivation of latent CMV, which may further contribute to host immune exhaustion, ultimately leading to a poor antitumor response. Pre-emptive anti-CMV medication may, therefore, serve as an adjunct therapy, preventing deterioration of the patient's immunity during treatment for cancer. The present study evaluated ganciclovir treatment's in vitro effects on CMV-associated T cell exhaustion and the antitumor capacity of cells from cancer patients. Methods Peripheral blood mononuclear cells from patients with various types of cancers were treated with or without ganciclovir. Cells were then analyzed for T cell subpopulations and activation markers using flow cytometry, and their antitumor capacity was tested after exposure to dendritic cell–A549 fusion cells. Results Following ganciclovir treatment, there was a redistribution of T cell subpopulations which was clearly distinct from the baseline pattern of T cell exhaustion. Treatment resulted in increases in early differentiated proportions, including naive (untreated control cells vs. ganciclovir treated cells, p + ( p + CD8 + ( p p p p Conclusion These findings demonstrate that ganciclovir treatment in vitro directly affects immune function by partially reconstituting T cell subsets and restoring the T cell antitumor response.

Parameters determining the efficacy of adoptive CD8 T-cell therapy of cytomegalovirus infection

Reactivation of latent cytomegalovirus (CMV) in the transient state of immunodeficiency after hematopoietic cell transplantation (HCT) is the most frequent and severe viral complication endangering leukemia therapy success. By infecting the bone marrow (BM) stroma of the transplantation recipient, CMV can directly interfere with BM repopulation by the transplanted donor-derived hematopoietic cells and thus delay immune reconstitution of the recipient. Cytopathogenic virus spread in tissues can result in CMV disease with multiple organ manifestations of which interstitial pneumonia is the most feared. There exists a 'window of risk' between hematoablative treatment and reconstitution of antiviral immunity after HCT, whereby timely reconstitution of antiviral CD8 T cells is a recognized positive prognostic parameter for the control of reactivated CMV infection and prevention of CMV disease. Supplementation of endogenous reconstitution by adoptive cell transfer of 'ready-to-go' effector and/or memory virus epitope-specific CD8 T cells is a therapeutic option to bridge the 'window of risk.' Preclinical research in murine models of CMV disease has been pivotal by providing 'proof of concept' for a benefit from CD8 T-cell therapy of HCT-associated CMV disease (reviewed in Holtappels et al. Med Microbiol Immunol 197:125-134, 2008). Here, we give an update of our previous review with focus on parameters that determine the efficacy of adoptive immunotherapy of CMV infection by antiviral CD8 T cells in the murine model.

Generation of Cytomegalovirus (CMV)-Specific CD4 and CD8 T Cell Lines Using Protein-Spanning Pools of pp65 and IE1 Derived Peptides.

BACKGROUND: Reactivation of latent CMV in immunocompromised recipients of allogeneic stem cell transplantation remains a major cause of morbidity and mortality. Reconstitution of immunity by CMV specific immunotherapy is an attractive alternative to drugs currently used, which show high toxicity and are sometimes ineffective. It has been demonstrated that CD4 helper T-cell function is crucial for the persistence of in vivo transferred CD8 CMV-specific CTL. Based on this finding, we have explored the feasibility of generating both anti-CMV CD4 and anti-CMV CD8 T-cell lines.METHODS: Dendritic Cells (DC) were generated from donor peripheral blood (PB) monocytes after a 7-day culture in the presence of GM-CSF plus IL-4 and matured with TNF-α, IFN-α, IFN-γ, IL1-β, POLI I:C. Matured-DC were then pulsed with a pool of 50 peptides spanning pp65 and IE1 proteins which are recognised by both CD4 and CD8 T lymphocytes. Donor T cells were stimulated three times at a T cell/DC ratio of 1:6 on day 0, +7 and +14 with mature peptide pulsed-DC. At the end of the culture the specificity of generated T cells was determined as percentage of pentamer-positive cells and intracellular IFN-γ production after incubation with peptide pulsed-DC. Cultured T cells were also analysed for their ability to proliferate in response to peptide pulsed-target cells, to kill them in a standard citotoxicity assay and to migrate in response to inflammatory (CXCL9, CCL3 and CCL5) and constitutive (CXCL12) chemokines.RESULTS: CMV-specific T cell lines were generated from five CMV seropositive donors. In four cases CD4 and CD8 CMV-specific T cell lines were expanded successfully. Cultured T cells expressed CD8 (mean= 70%, range 60–81%) and CD4 (mean= 20%, range 15–28%) and showed a CD45RA- CCR7- Effector Memory phenothype (mean=26%, range 19–30%) or a CD45RA+ CCR7- T Effector Memory RA-Positive phenothype (mean=67%, range 59–77%). An enriched CMV-specific T cell population was observed after staining with pentamers (7–45% pentamer-positive T cells). Furthermore, 90% of CD8+ and 40% of CD4+ T cells expressed high levels of intracytoplasmatic perforin and granzyme. In 4/5 cases tested, cutured T cells showed a cytolitic activity against CD8-peptide pulsed target cells (average lysis=50%, range 40–55%) and to a lesser extent against CD4-peptide pulsed target cells (average lysis=35%, range 30–40%). In addition, cultured T lymphocytes were able to proliferate and to produce intracytoplasmic IFN-γ (average production=50%, range 35–60%) after exposure to peptide-pulsed DC. Finally, Cultured T cells strongly migrated in response to chemokines (CXCL9, CCL3 and CCL5) involved in the recruitment of effector cells during viral infection.DISCUSSION: In conclusion, a great advantage of this method is represented by the possibility to generate anti-CMV CD4+ T cells, which could support in vivo the persistence of re-infused CMV-specific CTL. Moreover, the possibility of generating peptides under GMP conditions would facilitate the translation of this approach into clinical intervention.

Transcriptional activation of the promoter of human cytomegalovirus immediate early gene (CMV-IE) by the hepatitis B viral X protein (HBx) through the NF-κB site

The reactivation of latent cytomegalovirus (CMV) in a human by another viral infection may induce virus-related symptoms. Based on this presumption, we investigated the effect of HBx on the activation of the CMV-IE, which is also known as a transactivator and potential oncogene. The HBx transactivated the CMV-IE promoter by up to 4- and 18-fold factors in human liver HepG2 and lung fibroblast MRC-5 cells, respectively. Cotransfection of HBx with each transcription factor presented in the CMV-IE promoter showed that only NF-κB synergistically activated the promoter by up to a 14-fold factor. Serial deletion assays and point mutation analysis showed that the third NF-κB site (nt −267 to −258) and the second one (nt −162 to −153) appeared as the major responsible site and minor one, respectively, for the transactivation. These results suggest the possibility that the HBV infection of a cell previously infected by CMV would exert influence on the reactivation of the latent cytomegalovirus in a human to induce virus-related symptoms.

Zinc finger and carboxyl regions of adenovirus E1A 13S CR3 are important for transactivation of the cytomegalovirus major immediate early promoter by adenovirus.

Reactivation of latent cytomegalovirus (CMV) is an important cause of disease in susceptible patients. We previously demonstrated that an adenovirus early gene product can transactivate the CMV major immediate early (IE) promoter in inflammatory cells. This effect was due to the conserved region 3 (CR3) of the adenovirus E1A 13S gene product. There are two domains in the CR3 region, a zinc finger (aa 147-177) and a carboxyl (aa 180-188) domain. Both are crucial for transactivation of downstream promoter elements of adenovirus in E1A 13S. We sought to determine if either or both of these specific domains is also necessary for transactivation of the CMV IE promoter by the adenovirus E1A 13S gene product. We cotransfected T-lymphocyte Jurkat cells and monocyte/macrophage-like THP-1 cells with plasmids expressing wild-type (WT) or CR3 mutant E1A 13S and a CMV IE chloramphenicol acetyltransferase (CAT) reporter construct. With extracts of cells coinfected with E1A WT set to 100%, mutation in the zinc finger domain, the carboxyl domain, or both domains decreased CMV IE CAT activity by >/= 96%. In contrast, a mutation in the region between the zinc finger and carboxyl domains reduced CMV IE CAT activity by only 24 to 26%. Mixing studies in Jurkat cells confirmed the importance of these domains. We also evaluated the active site of the CMV IE promoter involved in transactivation in THP-1 cells using CMV IE promoter deletions and single promoter element constructs. These studies showed that progressive deletion of the 19-bp CMV IE repeats containing cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) sites resulted in progressive loss of activity. The importance of this element was confirmed using single promoter elements containing CMV IE 16-, 18-, 19-, and 21-bp repeats. Finally, using a 19-bp single promoter element construct and the CR3 mutants we demonstrated that mutations in the zinc finger (C171S) carboxyl region (S185N) or both regions (C171S/ S185N) resulted in significant (83, 94, and 85%) loss of activity. We conclude that the zinc finger and carboxyl domains of the CR3 region of E1A 13S are necessary for transactivation of the CMV promoter and that this occurs mainly through activation of the 19-bp CREB/ATF site of the promoter.

The use of computer-assisted video image analysis for the quantification of CD8 + T lymphocytes producing tumor necrosis factor α spots in response to peptide antigens

Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of single T lymphocytes forming cytokine spots after antigen contact in vitro. Herein computer-assisted video image analysis (CVIA) was applied to automatically determine the number and size of tumor necrosis factor α (TNF- α) spots formed by single blood-derived CD8 + T cells after contact with peptide-loaded target cells. With CVIA and TNF- α ELISPOT analysis we quantified CD8 + T cells responsive to HLA-A2.1-binding tyrosinase and influenza matrix peptides in healthy donors. We followed the course of the virus-specific T cell response in two HLA-A2-positive patients with reactivation of latent cytomegalovirus (CMV) infection during immunosuppressive therapy. The test proved sufficiently sensitive to detect in the blood of both patients a temporary expansion of CD8 + T lymphocytes reactive with a known immunogenic HLA-A2.1-binding peptide from glycoprotein B of CMV. Reactivity to peptide antigens was not only reflected by numeric increases of spot formation, but also by the appearance of larger spot areas, presumably formed by strongly peptide-reactive CD8 + T cells. We conclude that the combined use of the TNF- α ELISPOT assay and CVIA allows reliable monitoring of the T cell responsiveness to peptide antigens in peripheral blood.