Reconstitution of cytomegalovirus (CMV)-specific CD8 ؉ T cells is essential to the control of CMV infection in CMV-

It has been shown that activating killer Ig-like receptor (aKIR) genes are important for control of CMV reactivation after hematopoietic cell transplantation (HCT). To date, using the broad classification of KIR haplotypes A and B, the precise role of individual KIR genes in control of infection cannot be discerned. To address this, a consecutive case series of 211 non T-cell depleted HCT patients all at risk for CMV, were monitored bi-weekly for CMV DNA in plasma by Q-PCR and at intervals for CMV-specific T cell immunity. Comparing patients with CMV reactivation (n=152) to those with no reactivation (n=59), the presence of specific aKIR haplotypes in the donor, but not in the recipient, were associated with protection from CMV reactivation and control of peak plasma CMV DNA (p< 0.001). A donor aKIR profile, predictive for low risk of CMV reactivation, contained either aKIR2DS2 and aKIR2DS4 or had ≥ 5 aKIR genes. Neither donor nor recipient iKIR played a role in a protective effect. CD4+- and CD8+-specific CMV immunity did not explain reduced CMV infection. The initial control of CMV infection after HCT is managed by aKIR functions, and donor aKIR haplotypes deserve further evaluation in donor selection for optimized HCT outcome.

Late occurrence of cytomegalovirus (CMV) disease after day 100 after bone marrow transplantation has become an increasing problem; whether a quantitative measurement of CMV DNA in plasma by polymerase chain reaction (P-PCR) could be predictive of such disease was investigated. In a prospective study, 117 subjects undergoing allogeneic marrow transplantation were followed for 120 days with weekly CMV blood cultures, with day 35 bronchoalveolar lavage CMV cultures, with weekly CMV P-PCR, and with clinical follow-up for an additional 1 -2 years. Despite preemptive ganciclovir, CMV disease occurred in 9% of subjects, with a median time of onset of 176 days. Quantitative CMV P-PCR was associated with the late development of CMV disease (P Å .01). Of 43 subjects with positive P-PCR results, 23% developed CMV disease, but no disease occurred in the 74 subjects with negative P-PCR (P õ .001), despite the fact that 22% had CMV isolated from lung lavage fluid and 32% had CMV isolated from blood. Risk factors for the occurrence of human cytomegalovirusBMT, the presence of CMV DNA in plasma has been associated with risk for disease [3,4, 12] and pulmonary CMV (CMV) -related morbidity after allogeneic bone marrow transplantation (BMT) can be used to guide antiviral therapy [1 -infection [13], but the significance of the quantity of CMV DNA in plasma has not been studied. We evaluated whether 5]. The most important of these factors is the occurrence of asymptomatic CMV reactivation in blood or lung [2], and inquantitative plasma PCR is predictive of late CMV disease. fection of urine or throat has not been as predictive of later disease [1, 2]. In addition, the occurrence of graft-versus-host Methods disease (GVHD), and other factors predisposing to this, such as age ú20 years and HLA mismatch of donor and recipient, Study population. During 1993-1994are important risks for CMV infection and disease [6]. seropositive recipients of allogeneic BMT for hematologic malignancy or severe aplastic anemia were followed for the occurrence With the ability to determine CMV DNA in plasma by polyof CMV infection and disease in a prospective study. CMV infecmerase chain reaction (PCR) [7, 8] persons with CMV pulmonary infection [11]. In allogeneic No clinical decision was made on the basis of CMV P-PCR result. Thirty-six patients (31%) were treated because BAL fluid was positive for CMV; among the 81 BAL fluid CMV-negative subjects, an additional 35 were treated with ganciclovir for positive Received

The functional status of cytotoxic T lymphocyte (CTL) populations recognizing cytomegalovirus intermediate-early antigen (IE1) and pp65 polypeptides was investigated in peripheral blood mononuclear cells from hematopoietic stem-cell transplant (HSCT) and solid organ transplant recipients. Combined flow-based CD107a/b degranulation/mobilization and intracellular cytokine (ICC) assays using peptide libraries as antigens indicated that a significantly higher proportion of pp65-specific CTLs were in a more mature functional state, compared with IE1-specific CTLs. Degranulation/multiple cytokine ICC assays also indicated that a significantly higher proportion of pp65-specific than IE1-specific CTLs secreted both interferon- gamma and tumor necrosis factor- alpha and possessed greater cytotoxic potential. These results support our earlier findings of functional differences between CTLs recognizing individual epitopes within the IE1 and pp65 antigens in healthy donors and HSCT recipients and extend them to a broader array of human leukocyte antigen-restricted responses to those antigens. We also provide evidence of a relationship between cytotoxic function and the ability of cytomegalovirus-specific CTLs to secrete multiple cytokines.

In a prospective longitudinal study, detection of cytomegalovirus (CMV) DNA in plasma (plasma polymerase chain reaction [PCR]) was compared with PCR of CMV DNA in peripheral blood leukocytes (PBL PCR), the CMV pp65 antigenemia assay, and viral cultures from blood, urine, and throat of 29 patients, 14 of whom received pp65 antigenemia-guided early ganciclovir treatment and 15 of whom received ganciclovir at engraftment. Among 328 blood samples tested by all methods, PBL PCR was the most sensitive test, followed by the pp65 antigenemia assay, plasma PCR, and viremia. In the 14 patients who received pp65 antigenemia-guided early treatment, the incidence of PBL PCR, pp65 antigenemia, plasma PCR, and viremia before day 100 was 79%, 79%, 71%, and 27%, respectively, with a median day of onset of day 32, 42, 45, and 51, respectively. Nine patients (64%) became positive by PBL PCR, pp65 antigenemia, and plasma PCR. Of 15 patients who were treated with ganciclovir at engraftment, 12 (80%) became positive by PBL PCR, plasma PCR, and/or pp65 antigenemia while receiving ganciclovir; 3 (20%) had breakthrough infection with all three methods, including 2 with high-grade antigenemia (more than three positive cells in duplicate staining); none of these patients subsequently developed positive CMV cultures or disease. In 49 specimens, PBL PCR and/or pp65 antigenemia assay could not be performed because of insufficient neutrophil counts. In conclusion, the sensitivity of plasma PCR is significantly lower than that of PBL PCR but similar to that of the pp65 antigenemia assay. Plasma PCR may be particularly useful in clinical situations in which a less sensitive and possibly more specific assay is warranted or in which leukocyte counts are inadequate to perform cell-based assays.

The important role of activating Killer Immunoglobulin-like Receptors (aKIR) in protecting against cytomegalovirus (CMV) reactivation has been described previously in hematopoietic cell transplantation (HCT). More specifically, the presence of multiple aKIR and the presence of at least KIR2DS2 and KIR2DS4 in the donor genotype identified a group of HCT patients that were at low risk for CMV reactivation. However, CMV infection still occurs in patients with KIR protective genotype and the question was raised as to whether this was due to the lack of KIR expression. In this report, the expression of KIR2DS2 and 2DS4 gene, as measured by mRNA-based Q-PCR both in the donor cells and in the HCT recipient cells was studied relative to CMV reactivation. In the control samples from healthy HCT donors, the median range of for KIR2DS2 and KIR2DS4 expression was low with 35% considered null-expressers. Interestingly, KIR2DS2 and KIR2DS4 expression was elevated after HCT when compared to donor expression prior to transplant, and significantly elevated in the CMV viremic (V) compared to non-viremic (NV) HCT recipients. CMV seropositivity of donors was not associated with aKIR expression, and donor null-expression in those with KIR2DS2 or KIR2DS4 genotype did not predict for CMV reactivation in the recipient. After controlling for other transplant factors that included donor type (sibling or unrelated), transplant source -bone marrow (BM) or peripheral blood stem cells (PB) and acute GVHD grade, the result of the regression analysis of elevated KIR gene expression was found to be associated for both KIR2DS2 and KIR2DS4, with seven fold increase in risk for CMV reactivation. We speculate that the elevated aKIR expression in CMV viremic HCT recipients is either coincidental with factors that activate CMV or is initiated by CMV or cellular processes responsive to such CMV infection reactivation.

MSL-109 is a monoclonal antibody specific to the cytomegalovirus (CMV) glycoprotein H with high neutralizing capacity. In a prospective, randomized, double-blind study, allogeneic hematopoietic stem cell transplantation (HSCT) recipients with positive donor and/or recipient serology for CMV before transplantation received either 60 mg/kg MSL-109 (n = 59), 15 mg/kg MSL-109 (n = 60), or placebo (n = 60) intravenously every 2 weeks from day -1 until day 84 after transplantation. CMV pp65 antigenemia, CMV-DNA load in plasma, and viremia by culture were tested weekly. Primary end points were development of pp65 antigenemia at any level and/or viremia for which ganciclovir was given. There was no statistically significant difference in CMV pp65 antigenemia or viremia among patients in the 60-mg group (pp65 antigenemia, 47%; viremia, 15%), the 15-mg group (52%; 23%), and the placebo group (45%; 17%). There was also no difference in maximum levels of pp65 antigenemia, time to clearance of pp65 antigenemia after start of ganciclovir, CMV disease, invasive bacterial and fungal infections, time to neutrophil and platelet engraftment, acute graft-versus-host disease, days of hospitalization, and overall survival rate among the 3 groups. However, a subgroup analysis of CMV-seronegative recipients with a seropositive donor (D+/R-) showed a transiently improved survival rate by day 100 in MSL-109 recipients (mortality: 60-mg group, 1/13; 15-mg group, 1/12; placebo group, 6/10 [P = .02 for 60-mg versus placebo groups; P = .08 for 15-mg versus placebo groups]); by the end of follow-up, the difference was no longer statistically significant. The improved survival rate in D+/R- patients could not be attributed to a reduction in CMV disease; however, MSL-109 was associated with improved platelet engraftment and less grade III to IV acute graft-versus-host disease in this subgroup. In a subgroup analysis of CMV-seropositive recipients of MSL-109 (D+/R+ and D-/R+), overall mortality was increased compared to that of the placebo group (P = .12 for the 60-mg versus placebo groups, P = .05 for the 15-mg versus placebo groups, and P = .04 for the dose levels combined versus placebo). MSL-109 was well tolerated and no immune response to the drug was observed. Thus, MSL-109 was safe but did not reduce CMV infection in allogeneic HSCT recipients. The transient survival advantage seen early after transplantation in CMV D+/R- patients and the negative effect on survival in seropositive patients remain unexplained. Thus, there is no evidence that MSL-109 is beneficial in CMV-seropositive HSCT recipients.

A panel of 7 human cytomegalovirus (CMV) epitope peptides and corresponding major histocompatibility class 1 tetramers was used to evaluate cellular immunity in healthy seropositive donors and in hematopoietic stem-cell transplant recipients. Broad CMV-specific T cell responses to epitopes were found within several CMV polypeptides and were restricted by multiple human leukocyte antigen alleles. Their cytotoxic functionality was evaluated by use of an assay that measures transient surface levels of lysosomal membrane proteins LAMP-1 (CD107a) and LAMP-2 (CD107b) after peptide stimulation. This assay can be combined with tetramer staining of antigen-specific CD8(+) T lymphocytes and has potential as a surrogate marker for cytotoxic function. CD8(+) T lymphocytes specific for epitopes within the pp65 or pp50 gene products exhibited significantly higher functionality, compared with populations recognizing CMV major immediate early-1 epitopes. These functional differences between T lymphocyte populations within the same individual may have implications for protection against CMV.