Seronegative Donors Research Articles

Cytomegalovirus Reactivation Shapes NK Cells Diversity Following Allogeneic Hematopoietic Stem Cell Transplantation

Natural killer (NK) cells are the first lymphocyte population to reconstitute following allogeneic hematopoietic stem cell transplantation (HSCT) and are key players in immune defense against viral infections and malignant transformation. NK cell numbers generally recover within the first month post-transplant, but the acquisition of mature NK cell phenotype and full functional competency can take over 6 months and is influenced by various host and donor factors. Cytomegalovirus (CMV) infection has been shown to modulate NK cell maturation after HSCT.The diversity of the NK cell repertoire is dictated by a variety of combinatorially expressed activating and inhibitory receptors that dictate the NK activation status. Moreover, whereas the expression of inhibitory receptors is primarily genetically determined, environmental factors such as viral infections influence the expression of activating receptors to a great extent.. We propose that assessment of diversity could provide a different perspective for the evaluation of the NK cell compartment after HSCT, since it is a quantitative measure that takes into account both the number and evenness of the different NK subpopulations.To better understand the factors that influence NK cell recovery after cord blood (CB) transplant (CBT) and specifically the influence of cytomegalovirus (CMV) infection on NK cell maturity, we used 40-parameter mass cytometry (CyTOF) to interrogate the NK cell repertoire. A panel including 37 monoclonal antibodies was designed to recognize NK cells lineage markers and receptors as well as intracellular markers such as transcription factors and adaptor proteins. We first evaluated and compared the diversity of NK cells in 10 CB units and peripheral blood (PB) from 20 healthy donors. We then examined the diversity of NK cells before and after CBT in 22 serially collected blood samples from in 10 CBT recipients.NK cell diversity was significantly lower in CB (mean 574, range 417-891) compared to PB samples from healthy donors (mean 3792, range 1284-5079; P=0.001), indicating less diversification within the naive CB NK compartment. After CBT, NK cell diversity was lower at earlier time point (Day30) (mean 1129, range 428-1768) compared to PB from healthy donors; P=0.01. The diversity of NK cells increased gradually over time following CBT (Day 30 mean 1129 range 428-1768; Day 60, mean 1185, range 515-1864; 4 months, mean 1711 range 597-2640).We also compared the diversity of NK cells in the PB of healthy CMV seronegative (n=10) and seropositive adult donors (n=10). The diversity of NK cells was higher in CMV seropositive vs. CMV seronegative healthy donors (3887 vs 2473; P=0.04). This difference in NK diversity was even more pronounced within the KIR positive (mean 1701, range, 981-2152) compared to the KIR negative subset (mean 551, range 456-647; P=0.02), indicating that CMV infection increases the richness of mature NK cells. In keeping with these findings, CMV infection after CBT was associated with a significantly greater diversity of NK cells, especially within the KIR positive compartment (mean 604, range 207-1035) compared to the KIR negative subset (mean 283, 257-457; P=0.025). However, in CMV negative patients, we found no difference in diversity within the KIR positive and negative subsets (mean 1120 vs. 1366; P=0.28). Taken together, these data suggest that NK cell diversity reflects NK cells differentiation and maturation, and that CMV shapes NK cell diversity, especially within the KIR positive compartment.To further understand how CMV influences NK cells diversity, we examined the top 15 NK cell subsets and their distribution at multiple timepoints before and after CMV reactivation post-CBT. CMV infection post-CBT was associated with a significant change in the distribution of NK subsets within the KIR positive population, with the top 15 subsets prior to CMV reactivation being mostly replaced by the emergence of new subsets. In contrast, the top 15 subsets within the KIR negative NK population remained stable. These data suggest that CMV drives NK cell maturation by differentiating KIR positive NK cells.In summary, we used high-dimensional single-cell data to evaluate NK cell reconstitution following HSCT. These data can help us better understand the biology of NK cell recovery after HSCT and discover the functional significance of NK cell diversity in the setting of viral infections. DisclosuresChamplin:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties.

Peptide Vaccination Against Cytomegalovirus (CMV) Elicits Immunological and Clinical Responses after Allogeneic Stem Cell Transplantation Even from a CMV Seronegative Donor

Introduction: Patients receiving an allogeneic stem cell graft from a cytomegalovirus (CMV) seronegative donor are prone to CMV reactivation with high risk of disease and mortality. Therefore we initiated a clinical phase I trial with a novel vaccine designed by our group: a CMV phosphoprotein 65 (CMVpp65)-derived peptide in water-in-oil emulsion (Montanide™) plus administration of granulocyte-macrophage colony stimulating factor.Material and Methods: 10 patients after allogeneic stem cell transplantation received four vaccines s.c. at a biweekly interval. Patients were monitored for clinical course and CMVpp65 antigenemia. CMV-specific CD8+ and gamma/delta T cells were analyzed by multi-color flow cytometry. Neutralizing anti-CMV antibody assays were established and correlated to clinical parameters.Results: Peptide vaccination was well tolerated, no drug-related serious adverse events were detected. 7 of 9 patients with CMVpp65 antigenemia cleared the CMV with enduring response > 1.5 years after 4 vaccinations and are still free from antigenemia until present. 2 patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An up to 6-fold increase in frequency of both CMV-specific CD8+ T cells or Vdelta2- gamma/delta T cells with a maximum of 1.32% CMV-specific CD8+ T cells ex vivo without priming and 13.7% Vdelta2- gamma/delta T cells of all CD8+ T cells was detected in five patients. Also titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. Humoral and cellular immune responses correlated with clearance of the CMV load.Conclusion: Administration of CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing. DisclosuresKuball:Gadeta B.V.: Membership on an entity's Board of Directors or advisory committees. Wuchter:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Gilead: Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy. Greiner:BMS: Research Funding.

Prevention of Viral Infections after T Cell Depleted Allogeneic Stem Cell Transplantation By Infusion of Multi-Antigen Specific T Cell Products

Opportunistic viral infections and relapse are major complications in patients after T cell depleted allogeneic stem cell transplantation (TCD alloSCT). Since the application of unmodified donor lymphocyte infusion (DLI) early after alloSCT results in a high risk of graft versus host disease (GVHD), infusion of selected populations of virus-specific donor T cells can be an effective approach to safely restore anti-viral immunity early after alloSCT. As part of the EU FP7 consortium T Control, in this phase I/II study the feasibility and safety of the generation and administration of selected populations of donor-derived T cells targeting multiple antigens (Ag) is assessed. The multi Ag-specific T cell products contained T cells targeting cytomegalovirus (CMV), Epstein Bar virus (EBV) and adenovirus (AdV) as well as T cells targeting tumor associated Ag (TAA) and minor histocompatibility Ag (MiHA) to boost the graft versus leukemia (GVL) reactivity. To assess efficacy, in-vivo appearance or expansion of Ag-specific T cells, and the effect on viral reactivations and/or disease relapse was evaluated for 20 weeks after infusion until regular DLI was applied.HLA-A*02+ patients treated for a hematological malignancy with an HLA-matched TCD alloSCT from a CMV and/or EBV seropositive donor were included in this study. 6-8 weeks after alloSCT, T-cells directed against HLA-A*02-restricted peptides of CMV, EBV and AdV, and the TAA NY-eso, WT-1, RHAMM, PRAME and proteinase 3 were isolated using the reversible streptamer-nanobead technology (Juno) by cliniMACS selection out of the naïve and/or memory T cell compartment from 2*10^9 donor PBMC. Depending on the HLA-typing of the patient/donor additional streptamers targeting viral peptides in A*01, A*24, B*07 or B*08 were added to the selection procedure. In case of patient/donor MiHA disparity in the GvL direction, the HLA-A*0201/HA-1h streptamer was also added. This procedure allows purification of T cells under GMP conditions in 1 day.20 multi Ag-specific T cell products were generated of which 19 met the release criteria. These products consisted of 0.5-12*10^6 cells containing purities of 46,0-94,3% target Ag-specific CD8+ T cells within the T cell compartment. In all products CMV and/or EBVas well as AdV virus-specific memory T cells were isolated comprising 99% of the target Ag-specific CD8+ T cells, while the other 1% included the TAA and MiHA specificities or CMV specific T cells from seronegative donors. 17 products were administered without infusion-related complications or GVHD; 2 patients experienced GVHD before infusion and did not receive their product.Of the 14 patients evaluated at this stage, 13 completed the follow-up period until DLI and 1 patient died during follow-up. No product-related adverse events were reported. In all 5 CMV- patients and in 3/9 CMV+ patients no CMV reactivation and no expansion of CMV-specific T cells was observed. The other 6 CMV+ patients experienced CMV reactivations. In 2/6 patients who received the product from a CMV+ donor CMV-specific T cells were detected with tetramer analysis and CMV was cleared. From the other 4 reactivating patients with CMV- donors 3 had circulating CMV-specific T cells already at the moment of infusion, whereas in 1 patient CMV-specific T cells clearly expanded after infusion, resulting in viral clearance in all 4 patients.All 14 donors were EBV+. In 7 patients EBV reactivations were observed, which coincided in 2/7 patients with the appearance of EBV-specific T cells and subsequent clearance of the virus. In 4/7 patients EBV reactivation was cleared without clear expansion of EBV-specific T cells. However, 1 patient required treatment for an EBV-PTLD, although ultimately EBV-specific T cells appeared and EBV was cleared.In none of the patients AdV DNA loads were detected in the follow-up period, while in 1 patient expansion of AdV-specific T cells was observed.Expansion of TAA and MiHA-specific T cells could not be demonstrated in-vivo using tetramer staining. More sensitive techniques will be required to visualize these cells. 2 patients showed relapse of their malignancy before DLI infusion.In this clinical study, we have shown that the reversible streptamer technology based generation and adoptive transfer of donor-derived multi Ag-specific T cell products is feasible and safe and can be used as a strategy to prevent viral infections in the interval between TCD alloSCT and DLI. DisclosuresGermeroth:Juno Therapeutics: Employment.

Administration of Most Closely HLA-Matched Multivirus-Specific T Cells for the Treatment of EBV, CMV, AdV, HHV6, and BKV Post Allogeneic Hematopoietic Stem Cell Transplant

Viral infections remain a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with donor-derived virus-specific T cells (VSTs) has proven safe and effective for the prophylaxis and treatment of EBV, CMV, AdV, BKV and HHV-6 infections post-HSCT. However, broader application is restricted by the time taken to prepare patient-specific products and the lack of virus-specific T cell precursors in cord blood and seronegative donors. Thus, to assess whether 3rd party multivirus-directed VSTs could produce clinical benefit when administered as an "off the shelf" product to partially HLA-matched allogeneic HSCT recipients with drug-refractory BKV, EBV, CMV, AdV and/or HHV-6 infections we initiated a Phase II clinical trial (fixed cell dose of 2x107 VSTs/m2) using a bank of 59 VST lines generated from healthy, eligible donors.To date, 32 patients with one (n=28) or two (n=4) drug-refractory infections have been infused with lines matched at 1 to 6 HLA antigens. Fifteen patients received VSTs to treat CMV (including 3 cases of CMV colitis), 13 for BKV (11 for hemorrhagic cystitis, 2 for nephritis), 4 for AdV (including 1 case of pneumonia), 2 for HHV-6 (including 1 case of encephalitis) and 2 for EBV (including 1 case of EBV-PTLD). Twenty-one patients received just 1 infusion of cells, while 11 required 2 or 3 infusions for sustained benefit. Despite the HLA disparity between the VST lines and the recipients, de novo graft versus host disease (GVHD) occurred in only 1 subject, who developed Grade I skin GVHD that resolved with topical steroids. One additional patient had a flare of chronic skin GVHD coincident with tapering of immunosuppression and 2 patients developed transient fevers a few hours post-infusion, which spontaneously resolved within a couple of hours.Based on viral load, as measured by quantitative PCR, 3rd party VSTs successfully controlled active infections in 29 of 31 evaluable patients: CMV (9 CR, 5 PR, 1 NR); EBV (2 CR); AdV (2 CR, 1 PR, 1 NR); BKV (4 CR, 9 PR); and HHV-6 (1 PR). Of note, all 11 patients with BK hemorrhagic cystitis, 2 of 3 patients with CMV colitis, and the patients with AdV pneumonia and HHV-6 encephalitis, all had marked improvement or resolution of symptoms within 6 weeks following VST treatment. In more than 50% of responders (n=16) we detected an increase in the circulating frequency of VSTs post-infusion, which were confirmed to be 3rd party VST origin in 8 and remained detectable for up to 12 weeks post-infusion. The eventual decrease in 3rd party VSTs coincided with endogenous immune reconstitution in most patients.These results confirm the feasibility and safety of 3rd party multivirus-directed VSTs, administered as an "off the shelf" product to treat drug-refractory infections/disease. The infused cells were capable of expanding in vivo and proved clinically effective against refractory EBV, CMV, AdV, BKV and HHV-6 reactivation and virus associated disease. DisclosuresTzannou:ViraCyte LLC: Consultancy. Brenner:Viracyte: Equity Ownership; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Viracyte: Equity Ownership. Heslop:Cell Medica: Patents & Royalties: Licensing agreement EBV-specific T cells; Viracyte: Equity Ownership; Chimerix: Other: Endpoint adjudication committee; Celgene: Patents & Royalties, Research Funding. Leen:ViraCyte LLC: Equity Ownership, Patents & Royalties.

Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells Is a Safe Strategy to Prevent Viral Reactivation in Recipients of Allogeneic T Cell Depleted Stem Cell Transplant

Background: Allogeneic stem cell transplant (SCT) recipients suffer from a defective T cell mediated immunity causing potentially fatal reactivation of latent viruses. After T cell depleted SCT we have observed over 80% CMV reactivation and significant additional costs ($58-74k/patient). Despite aggressive monitoring and pre-emptive therapy, reactivation and/or positive CMV serology brings a significantly higher risk for non-relapse mortality (NRM). Adoptive transfer of ex vivo generated virus specific donor T cells is effective as a treatment of infection post-SCT but it has not been tested as a prophylaxis of early reactivation. Here in a Phase I study we transferred multi-virus specific T cells (MVSTs) immediately post SCT, targeting CMV, Ebstein-Barr virus (EBV), BK and adenovirus (Ad) as a novel strategy to prevent viral reactivation in the recipients of T cell depleted sibling HLA-matched SCT.Methods: Subjects were eligible if enrolled in HLA-matched T cell depleted transplant protocol (13-H-0144) and deemed at risk for CMV reactivation. MVST cells were manufactured from SCT sibling donors. Elutriated lymphocytes were stimulated with autologous dendritic cells (DCs) pulsed with seven overlapping peptide libraries (pepmixes) spanning the length of immunodominant proteins from CMV (pp65 and IE1), EBV (BZLF1 and EBNA1), BK (LT and VP1) and Ad5. Cultures were maintained in G-Rex flasks for 14 days in presence of IL-7, IL-15 and IL-2 (after 72hrs), tested for sterility, phenotype, potency and cryopreserved. MVST cells were thawed and administered intravenously as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used at the following dose levels: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT), (Grade IV GVHD or any other severe adverse even (SAE) deemed to be at least "probably" or "definitely" related to the MVST infusion. Patients were followed to day +100 post SCT for secondary outcomes, including efficacy (Figure) and immune reactivity (for donor/recipient pairs).Results: MVST cells recognized the majority of pepmixes, were polyfunctional and robustly proliferated in response the cognate antigens, but minimally against allogeneic targets- suggesting a limited ability to induce GVHD. CDR3 sequencing of T cell repertoire showed a significant reduction in diversity and a striking dominance of a limited number of clonotypes in the final MVSTs. Nine subjects were enrolled and treated with MVST cells. MVSTs were successfully generated for all subjects, meeting the release criteria. Median time from SCT to MVST administration was 16 days (range D +6 to +52 post-SCT). Two subjects received MVST after day +30 due to cardiac instability and scheduling. There were no immediate infusion-related adverse events or DLT by day 42. One subject in cohort II developed a self-limiting grade I cytokine release syndrome in the setting of low-level EBV reactivation. One patient (cohort 1) developed de novo grade III aGVHD post-MVST infusion. CMV reactivation post-MVST occurred in 4 out of 8 evaluable subjects (50%) who completed D+100 post-SCT vs. 45 out of 52 patients (50% vs 87%; p value=0.031) in a historical cohort of recipients of T cell depleted SCT. In all cases CMV reactivation occurred during treatment with high dose steroids. In two cases MVST were generated from CMV seronegative donors and showed minimal activity against pp65 and IE1. In eight evaluable subjects who reached D+100 post-SCT there was no EBV-related disease, but we saw self-limiting low level EBV replication in 6 out of 8 cases. There were no cases of BK or Ad-related disease or viremia. ELISPOT analysis at D+100 revealed robust reconstitution of anti-viral immunity in analyzed recipients (vs. donors, Figure, B)Conclusions: This is the first report demonstrating that it is safe and feasible to use adoptively transferred allo-MVST immediately post-SCT to rapidly reconstitute anti-viral immunity and ameliorate the detrimental impact of the early viral reactivation in SCT recipient. No DLTs were seen. MVSTs had a markedly reduced allo-reactivity and carried a minimal risk of GVHD. Our results also suggest efficacy of this strategy in reducing viral reactivation. A Phase II portion of this study is currently enrolling patients. [Display omitted] DisclosuresSabatino:Kite: Employment, Equity Ownership.

Survey of Institutional Policies for Provision of "CMV-Safe" Blood in Ontario.

Debate continues on whether leukoreduction alone (LR) is sufficiently similar to leukoreduced cellular products drawn from cytomegalovirus (CMV)-seronegative (SN) donors to minimize the risk of transfusion-transmitted CMV (TT-CMV). We sought to determine the policy, inventory, and practice landscape of the province for TT-CMV mitigation. A web-based survey was distributed to hospitals in Ontario by Canadian Blood Services to collect data on their policies with respect to TT-CMV prevention. TT-CMV mitigation practices varied by patient population, hospital size, and region. Smaller institutions remain committed to dual prevention, whereas academic hospitals favor a single-measure approach. Although smaller institutions attempt to align their policies with leadership sites, emulation is often inaccurate. The demands for SN products also appear to be significantly lower than the current screening practices of Canadian Blood Services. Standardization is lacking on practices to prevent TT-CMV. Although there are barriers to harmonizing practices, the apparent shift to policies acknowledging LR as a sufficient protection is likely to continue.

Serological Patterns and Molecular Characterization of Occult Hepatitis B Virus Infection among Blood Donors.

BackgroundHepatitis B infections, characterized by the presence of a viral genome without detectable hepatitis B surface antigen (HBsAg; Occult hepatitis B infection [OBI]), have been reported recently.ObjectivesWe performed serological and molecular characterization of OBI among blood donors at Jiangsu province blood center during years 2013 and 2014.MethodsAll donor samples were routinely screened by double enzyme-linked immunosorbent assay (ELISA) for hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), Treponema pallidum (TP), and alanine aminotransferase (ALT). Single-reactive, nonreactive, and ALT-elevated samples were pooled or resolved by nucleic acid testing (NAT). Seromarkers were examined in HBsAg-/DNA+ samples. After 1 to 12 months of follow up, seromarkers were screened again to verify OBI samples.ResultsWe studied 157119 samples from blood donors. A total of 154397 ELISA nonreactive donor samples were identified, and HBV DNA was detected in 81 samples; no samples were positive for HIV or HCV RNA. Hepatitis B virus viral loads in most donors were less than 20 - 200 IU/mL. There was only one HBsAg-positive sample. Eighty HBsAg-/DNA+ samples were evaluated further. Of these samples, 85% (68/80) were reactive for anti-HBc and 36.2% (29/800) were reactive for anti-HBc and anti-HBs; 11.3% (9/80) did not have any detectable serological markers. Twenty-nine donors were followed up. One was HBsAg ELISA positive, and of six seronegative donors, all had anti-HBc and anti-HBs, but were negative for DNA. Samples were HBV genotypes B, C and D. Mutations in the S region of HBV DNA included S114T, G119R, P120S, T125M, C139Y, T140I, C147W, T148A, A159V/G, E164D, V168A, and R169C.ConclusionsOverall, we found that OBI was rare, but that the prevalence of OBI was slightly higher in Jiangsu than in other areas of China.

Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans.

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression.

HIV P24 antigen among HIV antibody seronegative blood donors in Osogbo Osun State, South Western Nigeria

Background: Efforts to curb the spread of HIV transmission through transfusion of blood and its products is still a problem because of challenge in countries using antibody-based rapid methods to detect infection during window period. Transmission of HIV through infected blood and its products accounts for approximately 10% in African region.Methods: This study analyzed true negativity of HIV infection in blood donors screened by ELISA test based on p24 core antigen detection. Four hundred and eighty (480) blood donors initially negative for HIV antibody by rapid screening kit, Determine™ HIV-1/2 (Abbott Laboratory, IL, USA) and re-screened with Immuno Comb® II HIV 1 and 2 (Bispot kit PBS Organics and Israel 2005). The samples were further tested for the presence of HIV antibody and p24 HIV core antigen using ELISA kits (Genscreen TM ULTRA HIV Ag–Ab) following manufacturer’s instructions. All donors initially tested negative for Hepatitis B virus, Hepatitis C virus.Result: Two (0.42%) of 480 blood donors tested positive for the p24 HIV core antigen. The two positive donors for the p24 antigen had multiple sexual partners and recent sexually transmitted infections.Conclusion: The association of the HIV p24 antigen with blood donation was highly significant (p = 0.000) and pose a great risk to recipients if screening of blood donor is only carried out by HIV antibody detection.

Reconstitution of cytomegalovirus-specific T-cell response in allogeneic hematopoietic stem cell recipients: the contribution of six frequently recognized, virus-encoded ORFs.

The reactivation of human cytomegalovirus (HCMV) in immunosuppressed patients is associated with significant morbidity. Testing HCMV-specific T-cell responses can help determine which patients are at high risk of HCMV disease. We optimized selection of HCMV antigens for detection of T-cell response of patients after allogeneic hematopoietic stem cell transplantation (HSCT) with the aim of identifying patients with insufficient control of HCMV reactivation. T-cell immune response to HCMV was monitored in 30 patients during the first year after HSCT. The HSCT recipients were classified according to their anti-HCMV T-cell response and the presence of HCMV DNA in the blood. We observed an inverse relationship between the magnitude of HCMV-specific T-cell responses against CMV lysate, phosphoprotein (pp) 65, immediate early-1 (IE-1), UL36, and UL55, but not to US3 and US29 detected by interferon-gamma (IFNγ)- ELISPOT and the level of HCMV DNA in the blood of patients during the 30 days following sampling. The study has revealed that patients who received a graft from a seronegative donor have a lower T-cell response against HCMV and increased probability of HCMV reactivation in comparison to the patients who had received their graft from a seropositive donor. The individual peptide pools and native HCMV antigens were useful for monitoring the time course of the anti-HCMV response by IFNγ-ELISPOT, which proved to have a prognostic value. Besides widely employed peptide pools of pp65 and IE-1, the use of antigens UL36 and UL55, but not US3 or US29, increased sensitivity of the test.

Cytomegalovirus infection does not impact on survival or time to first treatment in patients with chronic lymphocytic leukemia.

Human cytomegalovirus (HCMV) is a widely prevalent herpes virus which establishes a state of chronic infection. The establishment of CMV‐specific immunity controls viral reactivation and leads to the accumulation of very large numbers of virus‐specific T cells which come to dominate the immune repertoire. There is concern that this may reduce the immune response to heterologous infections and HCMV infection has been associated with reduced survival in elderly people. Patients with chronic lymphocytic leukemia (B‐CLL) suffer from a state of immune suppression but have a paradoxical increase in the magnitude of the CMV‐specific T cell and humoral immune response. As such, there is now considerable interest in how CMV infection impacts on the clinical outcome of patients with B‐CLL. Utilizing a large prospective cohort of patients with B‐CLL (n = 347) we evaluated the relationship between HCMV seropositivity and patient outcome. HCMV seropositive patients had significantly worse overall survival than HCMV negative patients in univariate analysis (HR = 2.28, 95% CI: 1.34–3.88; P = 0.002). However, CMV seropositive patients were 4 years older than seronegative donors and this survival difference was lost in multivariate modeling adjusted for age and other validated prognostic markers (P = 0.34). No significant difference was found in multivariate modeling between HCMV positive and negative patients in relation to the time to first treatment (HR = 1.12, 95% CI: 0.68–1.84; P = 0.65). These findings in a second independent cohort of 236 B‐CLL patients were validated. In conclusion no evidence that HCMV impacts on the clinical outcome of patients with B‐CLL was found. Am. J. Hematol. 91:776–781, 2016. © 2016 Wiley Periodicals, Inc.

Impact of Donor and Recipient Cytomegalovirus Serostatus on Outcomes of Antithymocyte Globulin–Conditioned Hematopoietic Cell Transplantation

Although previous studies involving allogeneic hematopoietic cell transplantation (HCT) without in vivo T cell depletion by rabbit antithymocyte globulin (ATG) have reported a substantial survival difference between D−R− and D+R− patients, but little to no survival difference between D−R+ and D+R+ patients (D, donor; R, recipient; +, cytomegalovirus [CMV] seropositive; −, CMV seronegative), whether this applies to HCT using ATG is unknown. We studied 928 patients who underwent myeloablative HCT for hematologic malignancies in Alberta between 1999 and 2014 who received graft-versus-host disease (GVHD) prophylaxis using ATG (Thymoglobulin, 4.5 mg/kg) in addition to methotrexate and cyclosporine. D−R− and D+R− patients had similar survival (no significant difference). D−R+ patients had a substantially lower survival than D+R+ patients (41% versus 59% at 5 years; P = .001). This difference was attributed to higher nonrelapse mortality, apparently due to higher GVHD-associated mortality. Survival rates were also lower for D−R+ HLA-matched sibling transplant recipients compared with D+R+ HLA-matched unrelated donor transplant recipients (44% versus 66% at 5 years; P = .009). In conclusion, when using ATG, choosing a seronegative donor for a seronegative patient is relatively unimportant, whereas choosing a seropositive donor for a seropositive patient is important, even if this requires the use of a seropositive matched unrelated donor graft.

Primary Epstein-Barr virus infection, seroconversion, and post-transplant lymphoproliferative disorder in seronegative renal allograft recipients: a prospective cohort study.

Epstein-Barr virus (EBV)-seronegative renal transplant recipients are at risk of post-transplant lymphoproliferative disorder (PTLD). We compared primary EBV infection, seroconversion, and PTLD in EBV-seronegative patients who received renal allograft from seropositive or seronegative donors (D+/R- and D-/R-, respectively). We prospectively followed 25 D+/R- and 8 D-/R- recipients. We followed patients from January 1999 to June 2009 with clinical visits, monthly EBV polymerase chain reaction tests, and serologic tests for a period of 1 year after kidney transplantation and on an individual basis thereafter. Three patients (9%) developed PTLD including 2 early-onset (<12 months) and 1 late-onset (>12 months) disease. In D+/R- and D-/R- patients, the frequencies of PTLD (8% vs. 12.5%, P = 0.7), EBV seroconversion (64% vs. 50%, P = 0.4), and EBV viremia (40% vs. 25%, P = 0.6) were not significantly different. Clinical, serologic, and virologic surveillance as well as reduction in immunosuppression after evidence of primary EBV infection resulted in a PTLD rate of 9%, despite a seroconversion rate of 60.6%. Rate of graft loss after reduction in immunosuppression was 10% (2 of 20), which was not significantly different from 13 patients without EBV seroconversion (no graft loss, P = 0.5). Rates of viremia, seroconversion, and PTLD in D+/R- and D-/R- patients appear to be similar. The incidence of PTLD in renal transplants ranges from 0.5% to 2.9%. Our data show a significantly higher rate in EBV-seronegative renal allograft recipients, suggesting the need for close surveillance. Our data also suggest that donors for EBV-seronegative recipients may be accepted irrespective of positive or negative serostatus, with ongoing surveillance important in either circumstance.

IL-8 Alterations in HIV-1 Infected Children With Disease Progression.

Disease progression in HIV-1 infected children is faster than in adults. Less than 5% of the infected children maintain stable CD4 counts beyond 7 years of infection and are termed long-term nonprogressors (LTNPs). Delineating the host immune response in antiretroviral naïve (ART) and treated HIV-1 infected children at different disease stages will help in understanding the immunopathogenesis of the disease.A total of 79 asymptomatic, perinatally HIV-1 infected children (50 ART naïve and 29 ART treated) and 8 seronegative donors were recruited in this study. T- and B-cell activation PCR arrays were performed from the cDNA, using total RNA extracted from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1 infected children at different stages of the disease. The differentially expressed genes were identified. Quantitative RT-PCR was performed for the (interleukin-8) IL-8 gene and its transcriptional mediators, that is, SHP2, GRB2, and IL-8R (IL-8 receptor/CXCR1). Plasma levels of IL-8 were measured by flow cytometry.Gene array data revealed a higher expression of IL-8 in the ART naïve HIV-1 infected progressors and in ART nonresponders than LTNPs and ART responders, respectively. Quantitative RT-PCR analysis demonstrated a significant higher expression of IL-8 (P < 0.001), its receptor CXCR1 (P = 0.03) and the upstream signaling molecule SHP2 (P = 0.04) in the progressors versus LTNPs. Plasma levels of IL-8 were significantly higher in progressors versus LTNPs (P < 0.001), and ART nonresponders versus ART responders (P < 0.001). A significant negative correlation of plasma levels of IL-8 with CD4 counts (cells/μL) was observed in HIV-1 infected ART naïve subjects (r = -0.488; P < 0.001), while the IL-8 levels positively correlated with viral load in the ART treated children (r = 0.5494; P < 0.001). ART naïve progressors on follow up demonstrated a significant reduction in the mRNA expression (P = 0.05) and plasma levels of IL-8 (P = 0.05) post 6 months of ART initiation suggesting the beneficial role of ART therapy in reducing inflammation in infected children.Our data suggest that IL-8 may serve as a potential prognostic marker in adjunct with CD4 counts to monitor disease progression in the HIV-1 infected children and the efficacy of ART.

An optimized IFN-γ ELISpot assay using T-activated ® proteins for determination of CMV-specific cell-mediated immunity

Abstract In healthy individuals, Cytomegalovirus (CMV) infections are controlled by CMV-specific cell-mediated immunity (CMI). However, functional impairment of the CMI in immunocompromized individuals can lead to uncontrolled CMV-replication. Thus, monitoring CMV-specific CMI is highly relevant for the prognosis of CMV-associated diseases. Here we show the optimization and technical validation of a monitoring tool for the determination of CMV-specific CMI. Phenotype, quantity and cytokine expression profile of T cells from 19 CMV-seropositive healthy donors reactivated by CMV (IE-1, pp65) -specific pools of overlapping 15mer peptides and the corresponding T-activated proteins® were compared using a 10-parameter flow cytometry-based intracellular cytokine staining assay. Subsequently, an IFN-g ELISpot protocol using T-activated proteins® was established and validated. Pp65 peptide pool stimulation revealed significant higher CD8+ T-cell responses as the corresponding T-activated proteins® (p=0.006). CD4+ T-cell responses however were comparable. Testing of T-activated proteins® in seronegative donors revealed a specificity of 100%. Use of T-activated proteins® with the optimized IFN-g ELISpot resulted in coefficients of variation for inter-assay, inter-operator and inter-site variations of less than 22 %. A linear correlation between the amount of CMV protein-reactive cells and total PBMC counts was observed. Given the natural antigen processing of T-activated proteins® along the cross-presentation pathway, reflecting the in vivo situation, these proteins in combination with the optimized IFN-g ELISpot serve as a valuable tool to monitor the CMV-specific CMI.

Impact of Donor Epstein-Barr Virus Serostatus on the Incidence of Graft-Versus-Host Disease in Patients With Acute Leukemia After Hematopoietic Stem-Cell Transplantation: A Study From the Acute Leukemia and Infectious Diseases Working Parties of the European Society for Blood and Marrow Transplantation.

We investigated the effect of Epstein-Barr virus (EBV) serostatus on the overall outcome of allogeneic hematopoietic stem-cell transplantation (allo-HSCT). The study included 11,364 patients who underwent allogeneic peripheral-blood or bone marrow transplantation for acute leukemia between 1997 and 2012. We analyzed the impact of donor and recipient EBV serologic status on overall survival, relapse-free survival, relapse incidence, nonrelapse mortality, and incidence of graft-versus-host disease (GVHD) after allo-HSCT. Patients receiving grafts from EBV-seropositive donors had the same overall survival as patients who received grafts from EBV-seronegative donors (hazard ratio [HR], 1.05; 95% CI, 0.97 to 1.12; P = .23). Seropositive donors also had no influence on relapse-free survival (HR, 1.04; 95% CI, 0.97 to 1.11; P = 0.31), relapse incidence (HR, 1.03; 95% CI, 0.94 to 1.12; P = .58), and nonrelapse mortality (HR, 1.05; 95% CI, 0.94 to 1.17; P = .37). However, in univariate analysis, recipients receiving grafts from seropositive donors had a higher risk of chronic GVHD than those with seronegative donors (40.8% v 31.0%, respectively; P < .001; HR, 1.42; 95% CI, 1.30 to 1.56). When adjusting for confounders, higher risk was identified for both acute and chronic GVHD. In seronegative patients with seropositive donors, the HR for chronic GVHD was 1.30 (95% CI, 1.06 to 1.59; P = .039). In seropositive patients with seropositive donors, the HR was 1.24 (95% CI, 1.07 to 1.45; P = .016) for acute GVHD and 1.43 (95% CI, 1.23 to 1.67; P < .001) for chronic GVHD. Seropositive patients with seronegative donors did not have an increased risk of GVHD. Our data suggest that donor EBV status significantly influences development of acute and chronic GVHD after allo-HSCT.

Evolution of human cytomegalovirus-seronegative donor/-seropositive recipient high-risk combination frequency in allogeneic hematopoietic stem cell transplantations at Institute of Hematology and Blood Transfusion during 1995-2014.

Human cytomegalovirus (HCMV) establishes lifelong latent infection that can result in severe life-threatening disease in immunosuppressed patients after hematopoietic stem cell transplantation (HSCT). An HCMV-seropositive transplant recipient who receives a graft from a seronegative donor (R+/D-) is at high risk of recurrent HCMV reactivation. To assess the incidence of R+/D- combination, we retrospectively evaluated HCMV-seronegative donors for 746 allogeneic HSCT treatments carried out at our center during 1995-2014. In our cohort, 20% HCMV-seronegative HSCT recipients, 21% HCMV-seronegative related graft donors, and 52% HCMV-seronegative unrelated graft donors were included. Analyses of the HCMV serostatus of hematopoietic stem cell donors during 2 consecutive calendar periods (1995-2005 and 2006-2014) showed a significant increase in the proportion of seronegative donors (odds ratio [OR]=1.947). In addition, the number of HSCT treatments using an unrelated donor increased (OR=2.376). Finally, the use of grafts from countries with a very low HCMV prevalence increased. This increase in HCMV seronegativity in unrelated donors and the increased proportion of unrelated donors were responsible for the increased occurrence of the high-risk combination R+/D- (OR=1.680). If the reduction in the rate of HCMV-seropositive graft donors continues, an increased frequency of HCMV reactivation events in our transplant recipients can be expected, because of the increasing occurrence of the high-risk R+/D- combination.

Significance of nucleic acid testing in window period donations: Revisiting transfusion safety in high prevalence-low resource settings

Background: Safe blood transfusion to the people is essential requirement of health care delivery system. Despite the mandatory screening of blood with newest, very sensitive serological test, considerable risk remains for transfusion transmission of virus due to window period infections. Study was aimed to analyze the efficacy of Mini pool Nucleic Acid Amplification Testing as additional donor screening program and its role in improving blood safety in the high prevalence population. Material and Methods: Study was performed at a tertiary-care, accreditated hospital from June 2013 to December 2015 All negative cases for anti-HIV, anti-HCV and HBsAg by ELISA were subjected to MP-NAT to detect HIV-1, HIV-2 and HCV-RNA with HBV DNA.Results: In 31 months, 20470 donations were received of which whole blood donations were 16997 (83.03%) and 3473 (16.97%) apheresis. Out of 16997 donations, 446 (2.61%) were seroreactive. Out of 16551 sero-negative donors subjected to MP-NAT testing, 17 (0.10%) were NAT reactive (NAT yield -1 in 974). Out of 17 cases, 12 were HBV (1 in 1379), four HCV (1 in 4138) and one HIV NAT reactive (1 in 16551). Conclusion: NAT has improvised the blood safety by detecting the virus in the pre-seroconversion, window period thereby providing much higher sensitivity as compared to newest generation serological tests. In countries with high incidence of infection with significant number of window period donations, NAT can serve as a valuable tool along with other serological testing in high prevalence, resource constrained countries to achieve the goal of zero risk of blood.

Interaction of non-human primate complement and antibodies with hypermucoviscous Klebsiella pneumoniae.

Emergent hypermucoviscosity (HMV) phenotypes of Klebsiella pneumoniae have been associated with increased invasiveness and pathogenicity in primates. In this study, we investigated the interaction of African green monkeys (AGM) (Chlorocebus aethiops sabaeus) complement and antibody with HMV and non-HMV isolates as in vitro models of primate infection. Significantly greater survival of HMV isolates was evident after incubation in normal serum or whole blood (p < 0.05) of AGM donors when compared to non-HMV strains. Greater survival of HMV strains (p < 0.05) was found after incubation in whole blood and serum from seropositive donors when compared to seronegative donor samples. Additionally, significantly greater amounts of K. pneumoniae were phagocytozed by AGM leukocytes when complement was active (p < 0.05), but no difference in uptake was observed when serum from seropositive or seronegative animals was used in challenged cells utilizing flow cytometry. Results demonstrate that interaction of cellular and humoral immune elements play a role in the in vitro killing of K. pneumoniae, particularly HMV isolates. Neither AGM serum, nor washed whole blood effectively killed HMV isolates; however, assays using heparinized whole blood of seronegative donors significantly reduced viability of HMV and non-HMV strains. The lack of bacterial killing observed in seropositive donors treatments could be at least partially associated with low IgG2 present in these animals. A better understanding of the pathogenesis of klebsiellosis in primates and host immune response is necessary to identify surface molecules that can induce both opsonizing and bactericidal antibody facilitating killing of Klebsiella, and the development of vaccines in human and animals.

The natural history of recent hepatitis C virus infection among blood donors and injection drug users in the country of Georgia.

IntroductionHepatitis C virus (HCV) infection is a serious health problem in Georgia.MethodsWe conducted a prospective study to identify and characterize the natural history of recent HCV infection since very first days of infection. Recent HCV infection was defined as detectable plasma HCV RNA in the absence of anti-HCV antibodies.ResultsA total of 7600 HCV seronegative blood donors and 3600 HCV seronegative drug users were screened for recent HCV infection. Among them 7 (0.09 %) blood donors and 10 (0.28 %) drug users tested positive for HCV RNA and were classified as having recent HCV infection. Of these 17 patients 4 (23.5 %) spontaneously cleared the virus by the end of 24 week follow-up. Five clinical forms of recent HCV infection were identified during the follow-up. Four patients had symptomatic disease, including 3 patients with jaundice and other clinical symptoms (2 of them cleared virus) and 1 patient only had other symptoms without jaundice. All symptomatic patients had ALT elevation. Three distinct variants of asymptomatic disease were identified in 13 patients: 9 patients had ALT elevation and none cleared the virus; 2 patients developed chronic disease without ALT elevation; 2 patients cleared virus without anti-HCV seroconversion and without ALT elevation; this form can be described as transitory HCV viremia.ConclusionAdditional studies are needed to define clinical and public health implications of transitory HCV viremia. Our study suggests the need for implementing nucleic acid testing of blood donors and key populations in order to more effectively identify HCV infected persons.