Role of pentamer complex-specific and IgG subclass 3 antibodies in HCMV hyperimmunoglobulin and standard intravenous IgG preparations.

BACKGROUND: HCMV hyperimmunoglobulin-preparations (HIG) contain high concentrations of HCMV-specific IgG. The reduced maternofetal-HCMV-transmission rate of IgG may be due to HCMV-specific neutralizing antibodies against the HCMV pentameric complex (PC). In contrast to HIG, standard intravenous immunoglobulin (IVIG) may have more neutralization (NT) capacity than HIG due to higher IgG subclass 3 levels (Planitzer et al., 2011). METHODS: We investigated the HCMV-specific NT-capacity of HIG Cytotect®, using a recombinant pentameric complex (gHgLUL128-131A) for specific antibody-depletion. We used a modified UL130-peptide (TANQNPSPPWSKLTYSKPH) based on original-sequence of Saccoccio et al. (Vaccine 29(15):2705-2711, 2011) (SWSTLTANQNPSPPWSKLTY) as neutralization target. Both UL130-peptides and the PC were bound via sixfold HisTag and anti-HisTag mAbs to magnetic beads to deplete HCMV-specific IgGs from HIG (Cytotect®). Modifying this depletion strategy, we analyzed the role of IgG subclass 3 in both HIG and IVIG. RESULTS: After CMV IgG-normalization of HIG and IVIG, we found a significant trend towards a decrease (16%) of neutralization-capacity for the UL130 TAN-peptide, but not for the original UL130 SWS-peptide. However, highly significant loss of NT-capacity could be only observed by PC depletion (42%). The IgG subclass 3 depletion revealed no significant reduction of NT-capacity in both HIG and IVIG. CONCLUSION: Via specific antibody depletion, we could demonstrate that pentameric complex-specific antibodies are present in HIG and bind to the recombinant PC resulting in a highly significant reduction of NT-capacity compared to the UL130 TAN-and SWS-peptides. We could not confirm the functional role of IgG subclass 3 neutralizing antibodies in IgG-preparations.

Case report: severe cytomegalovirus primary infection in an immunocompetent adult with disseminated intravascular coagulation treated with valganciclovir.

BACKGROUND: Disseminated intravascular coagulation (DIC) is a very rare complication of disseminated cytomegalovirus (CMV) infection. So far it is mainly described for immunocompromised patients. CASE PRESENTATION: A 49-year-old immunocompetent Caucasian male presented with sudden onset of fever and DIC due to primary CMV infection, which was treated with Valganciclovir. CMV-specific IgG-avidity and epithelial cell-specific neutralisation-capacity developed five weeks after onset of symptoms. We describe the first case of an immunocompetent patient suffering from DIC due to a CMV primary infection successfully treated with Valganciclovir. CONCLUSIONS: Primary CMV infection can occur accompanied with life threatening complications even in immunocompetent patients. Immediate treatment with Valganciclovir should be considered as an early treatment of choice in severe cases since specific neutralisation capacity might need several weeks to develop.

Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL) and proteinuria predict severity of acute kidney injury in Puumala virus infection.

BACKGROUND: Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome (HFRS) that is caused by the Puumala virus. Periodic outbreaks have been described in endemic areas, with a substantial number of previously healthy individuals developing acute kidney injury (AKI). There is a considerable diversity in the clinical course of the disease, and few patients require renal replacement therapy. METHODS: We tested whether urinary neutrophil gelatinase associated lipocalin (uNGAL), urine albumin/creatinine ratio (uACR), urine protein/creatinine ratio (uPCR), urine dipstick protein, C-reactive protein, procalcitonin, leukocyte and platelet count, determined on admission to the hospital, can predict the severity of AKI. Sixty-one patients were analyzed during admission in the emergency department. RESULTS: The variables most strongly associated with peak plasma creatinine concentration were uNGAL (ß = 0.70, p <0.0001), uPCR (ß = 0.64, p = 0.001), uACR (ß = 0.61, p = 0.002), and dipstick proteinuria (ß = 0.34, p = 0.008). The highest AUC-ROC to predict stage 3 AKI according to the acute kidney injury network's (AKIN) classification was seen for uNGAL (0.81, p = 0.001). CONCLUSION: uNGAL accurately predicts the severity of AKI in NE. This could help emergency room physicians predict disease severity and allow for initial risk stratification.

Hallmark features of immunosenescence are absent in familial longevity.

Seropositivity for CMV is one of the parameters of the "immune risk profile" associated with mortality in longitudinal studies of the very elderly and may accelerate immunosenescence. Thus, any genetic factors influencing human longevity may be associated with susceptibility to CMV and CMV-accelerated immunosenescence. To test this, we analyzed long-lived families in the Leiden Longevity Study (LLS) in which offspring enjoy a 30% reduced standardized mortality rate, possibly owing to genetic enrichment. Serum C-reactive protein levels and the frequency of different T cell subsets were compared between 97 LLS offspring and 97 controls (their partners, representing the normal population). We also determined the capacity of T cells to respond against immunodominant Ags from CMV in a smaller group of LLS subjects and controls. CMV infection was strongly associated with an age-related reduction in the frequency of naive T cells and an accumulation of CD45RA-re-expressing and late-differentiated effector memory T cells in the general population, but not in members of long-lived families. The latter also had significantly lower C-reactive protein levels, indicating a lower proinflammatory status compared with CMV-infected controls. Finally, T cells from a higher proportion of offspring mounted a proliferative response against CMV Ags, which was also of greater magnitude and broader specificity than controls. Our data suggest that these rare individuals genetically enriched for longevity are less susceptible to the characteristic CMV-associated age-driven immune alterations commonly considered to be hallmarks of immunosenescence, which might reflect better immunological control of the virus and contribute to their decreased mortality rate.

CD209/DC-SIGN mediates efficient infection of monocyte-derived dendritic cells by clinical adenovirus 2C isolates in the presence of bovine lactoferrin.

Adenovirus often causes respiratory infection in immunocompromised patients, but relevant attachment receptors have largely not been defined. We show that the antiviral protein bovine lactoferrin enhances infection of monocyte-derived dendritic cells (MDDC) by adenovirus species C serotype 2 (2C) isolates. Under the same conditions infection of MDDC by human( )cytomegalovirus was reduced. Adenoviral infection was prominently enhanced by bovine but not human lactoferrin, and was not prominently enhanced using blood monocyte-derived macrophages, suggesting that the relevant receptor is expressed on MDDC. Infection of MDDC in the presence of bovine lactoferrin was blocked by mannan, and an antibody to CD209/DC-SIGN but not isotype control or CD46 antibodies. Lastly, U937 macrophages ectopically expressing CD209/DC-SIGN, but not parental U937 cells, were efficiently infected by adenovirus 2C in the presence of bovine lactoferrin. These results may provide a tool, given the high efficiency of infection, to dissect responses by myeloid cells to clinical adenovirus isolates.

UL74 of human cytomegalovirus reduces the inhibitory effect of gH-specific and gB-specific antibodies.

The human cytomegalovirus (HCMV) glycoproteins gH (UL75) and gL (UL115) can form complexes with gO (UL74) or with proteins of the UL128-UL131A locus. Deletion of gO abolishes cell-free virus transmission and renders cell-associated virus transmission in fibroblasts more sensitive to inhibition by human anti-HCMV serum. To test whether the latter effect is specific for gO, we compared mutants with deletions in UL74, UL99 and the UL128-131A locus regarding their sensitivity to anti-HCMV antibodies. UL74 deletion mutants were more sensitive to a further restriction by polyspecific or gH-specific antibodies than control mutants, showing that gO specifically protects focal growth against inhibitory antibodies. This effect was not confined to gH-specific antibodies, as UL74 deletion mutants were also inhibited by an anti-gB antibody. In conclusion, gO specifically promotes focal spread in the presence of gH and gB antibodies, thus contributing to the ability of HCMV to resist the host's immune response.

Cytomegalovirus reactivation and associated outcome of critically ill patients with severe sepsis.

INTRODUCTION: Sepsis has been identified as a risk factor for human cytomegalovirus (CMV) reactivation in critically ill patients. However, the contribution of CMV reactivation on morbidity and mortality is still controversial. Therefore, we analyzed the incidence and impact of CMV reactivation on outcome in patients with severe sepsis. METHODS: In a prospective longitudinal double-blinded observational study, 97 adult nonimmunosuppressed CMV-seropositive patients with new onset of severe sepsis were included. Leukocytes, plasma and tracheal secretions were examined weekly for CMV-DNA by PCR. Tracheal secretions were additionally tested for HSV (Herpes Simplex Virus)-DNA. The influence of CMV-reactivation on the endpoints was analysed by Cox proportional-hazard regression analysis. Time-dependency was evaluated by landmark analysis. RESULTS: Six out 97 died and five were discharged from the hospital within 72 hours and were excluded of the analysis. CMV reactivation occurred in 35 of the 86 (40.69%) analysed patients. HSV infection occurred in 23 of the 35 (65.7%) CMV reactivators. In 10 patients CMV-plasma-DNAemia appeared with a DNA-content below 600 copies/ml in four cases and a peak amount of 2,830 copies/ml on average. In patients with and without CMV reactivation mortality rates were similar (37.1% vs. 35.3%, P = 0.861), respectively. However, in the multivariate COX regression analyses CMV reactivation was independently associated with increased length of stay in the ICU (30.0, interquartile range 14 to 48 vs. 12.0, interquartile range 7 to 19 days; HR (hazard ratio) 3.365; 95% CI (confidence interval) 1.233 to 9.183, P = 0.018) and in the hospital (33.0, interquartile range 24 to 62 vs. 16.0, interquartile range 10 to 24 days, HR 3.3, 95% CI 1.78 to 6.25, P < 0.001) as well as prolonged mechanical ventilation (22.0, interquartile range 6 to 36 vs. 7.5, interquartile range 5 to 15.5 days; HR 2.6,CI 95% 1.39 to 4.94; P < 0.001) and impaired pulmonary gas exchange (six days, interquartile range 1 to 17, vs. three, interquartile range 1 to 7, days in reactivators vs. non-reactivators, P = 0.038). HSV reactivation proved not to be a risk factor for these adverse effects. CONCLUSIONS: These data indicate an independent correlation between CMV reactivation and increased morbidity in the well-defined group of nonimmunosuppressed patients with severe sepsis, but CMV reactivation had no impact on mortality in this group with low CMV-DNA plasma levels. Thus, the potential harms and benefits of antiviral treatment have to be weighed cautiously in patients with severe sepsis or septic shock.

The role of cytomegalovirus infection and disease in pediatric bone marrow transplant recipients in Mexico City in the context of viral drug resistance.

We aimed to identify those pediatric patients undergoing ABMT with CMV EOD who developed GCV resistance. Forty-seven patients were analyzed following ABMT. Prospective post-transplant CMV monitoring was performed weekly for the detection of viral leukocyte DNAaemia, viral plasma DNAaemia, and viral DNAuria by PCR. Plasma DNAaemia was confirmed from whole blood by the detection of CMV pp67 late mRNA using NASBA technology. In the cases of persistence of viral DNA in plasma, and positive viral RNA detection in blood, CMV drug resistance screening by comprehensive PCR-based RFLP and sequencing of the viral UL97 gene were performed retrospectively. Thirty of the 47 (63.82%) patients showed active CMV infection with 27/30 (74.4%) patients belonging to the D+R+ group and 25/30 with proven viral replication. In total, 2/30 (6.6%) children developed CMV pneumonia proven by immunohistochemistry. Screening of the viral UL97 gene revealed in one of these two cases (1/30, 3.3%) the simultaneous presence of two point mutations in codon 460 (M460V, M460I) conferring GCV resistance. The CMV seroprevalence (81%) and the incidence of active infection (63.8%) in Mexican children undergoing ABMT are very high.

Dendritic cells are susceptible to infection with wild-type adenovirus, inducing a differentiation arrest in precursor cells and inducing a strong T-cell stimulation.

Adenovirus infection after stem cell transplantation is a significant cause of morbidity and mortality, especially in children. A robust T-cell response induced by dendritic cells (DC) is crucial for clearing the virus, suggesting their pivotal role for the response to human adenoviruses (HAdV). Despite the widespread use of adenoviral vectors, the properties and kinetics of HAdV infection of DC have not been addressed yet. We show that a recent clinical HAdV, subgenus C/serotype 2 (strain BB2000-61), infects cells of the myeloid lineage. Infected DC produce early and late viral antigens and show an altered expression of surface markers. Infection of monocytes renders them refractory to differentiation into DC. Additionally, HAdV-infected DC are strong stimulators of CD8+ T cells. In summary, HAdV seems to manipulate the immune response by infection of DC and possibly uses the infection of monocytes as a means to escape recognition by T cells.

Dendritic cell vaccination in an allogeneic stem cell recipient receiving a transplant from a human cytomegalovirus (HCMV)-seronegative donor: induction of a HCMV-specific T(helper) cell response.

BACKGROUND AIMS: In the absence of a protective immune response, human cytomegalovirus (HCMV) infection remains a life-threatening complication after allogeneic stem cell transplantation (SCT), especially in recipients of grafts from HCMV-seronegative donors. After allogeneic SCT from a seronegative donor, prolonged and severe immune deficiency often leads to infectious complications. Vaccination with antigen-loaded dendritic cells (DC) has been shown to be a potent approach for the induction of antigen-specific cytotoxic T-cell responses in vivo. For protection from subsequent HCMV reactivation, a sustained immune response is necessary, including antigen-specific CD4(+) T cells. METHODS: We report the case of an 18-year-old girl with high-risk acute lymphoblastic leukemia that received an allogeneic SCT in CR2. After an HCMV infection, the graft was rejected and she received a second transplant from an HLA-mismatched, HCMV-seronegative family donor. She was treated with pp65-pulsed monocyte-derived DC at day 200 post-SCT, using a recombinant pp65 protein. Until day 200 post-SCT, HCMV reactivated six times with emerging viral resistance to antiviral chemotherapy. RESULTS: After vaccination with protein-pulsed DC, an induction and expansion of HCMV-specific T(helper) cells and cytotoxic T lymphocytes was observed, associated with a sustained clearance of the HCMV viremia. Antiviral treatment could be tapered without recurrence of viremia within the first year post-SCT. CONCLUSIONS: pp65-pulsed DC could induce antigen-specific T-cell responses even after a SCT from an HCMV-seronegative donor. After vaccination with pp65-pulsed DC, a sustained antigen-specific T-cell response prevented concurrent HCMV viremia. Emergence of antigen-specific T(helper) cells may be essential for a sustained, functional T-cell response post-SCT.

In vivo Downregulation of MHC Class I Molecules by HCMV Occurs During All Phases of Viral Replication but Is Not Always Complete.

Based on cell culture data, MHC class I downregulation by HCMV on infected cells has been suggested as a means of immune evasion by this virus. In order to address this issue in vivo, an immunohistochemical analysis of tissue sections from biopsy and autopsy materials of HCMV infected organs was performed. HCMV antigens from the immediate early, early, and late phase of viral replication, and cellular MHC class I molecules were detected simultaneously or in serial sections by immuno-peroxidase and immuno-alkaline phosphatase techniques. Investigated organs included lung, gastrointestinal tract, and placenta. Colocalization of MHC molecules with sites of viral replication as well as MHC expression in individual infected cells were analyzed. To detect immune effector cells at sites of viral replication, leukocytes, CD8+ lymphocytes, and HCMV antigens were stained in serial sections. While strong MHC class I expression was detected in the cells surrounding infected cells, it appeared downregulated in the majority of infected cells themselves, particularly in the late replication phase. Despite significantly reduced MHC class I signals on infected cells, sites of infection were infiltrated by inflammatory cells that consisted predominantly of CD8+ lymphocytes. The extent of inflammatory infiltrates was negatively correlated with the extent of HCMV infected cells. Taken together, our findings indicate that HCMV can downmodulate MHC class I expression in vivo, whereas cytokines originating from infiltrating immune effector cells probably up regulates MHC class I expression in noninfected bystander cells. The presence of cytotoxic lymphocytes in close contact to infected cells may reflect control of viral spread by these cells despite MHC class I downmodulation.

UL74 of human cytomegalovirus contributes to virus release by promoting secondary envelopment of virions.

The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC.

Effects of different CMV-heat-inactivation-methods on growth factors in human breast milk.

Preterm infants can inoculate virulent cytomegalovirus (CMV) through their mothers' raw breast milk. Complete virus inactivation is achieved only by heat treatment, but the effect on growth factors has never been assessed systematically. Insulin-like-growth-factor-1-, IGF-2-, insulin-like-growth-factor-binding-protein-2-, and IGFBP-3-concentrations were measured, before and after heating, in 51 breast-milk-samples from 28 mothers, and epidermal-growth-factor-concentrations in a subgroup of 35 samples from 22 mothers. Two heating methods were applied: Short-term (5 s) pasteurisation at 62, 65, and 72 degrees C, and long-term Holder-Pasteurisation (30 min) at 63 degrees C. IGF-1, IGF-2, IGFBP-2, and IGFBP-3 were measured by RIA, and EGF by ELISA. Heating for 30 min decreased significantly IGF-1 by 39.4%, IGF-2 by 9.9%, IGFBP-2 by 19.1%, and IGFBP-3 by 7.0%. In contrast, IGF-1, IGF-2, IGFBP-2, and IGFBP-3 were not altered significantly when using a short heating duration of 5 s, irrespective of the level of temperature, except for IGF-2 at 62 degrees C for 5 s (p = 0.041) and IGFBP-2 at 72 degrees C for 5 s (p = 0.025). Neither long- nor short-time heating methods changed the concentration of EGF. Only short heating methods (5 s, 62-72 degrees C) can preserve, almost completely, the concentrations of IGFs in human milk, whereas Holder-Pasteurization does not.

Rapid semiquantitative real-time PCR for the detection of human cytomegalovirus UL97 mutations conferring ganciclovir resistance.

BACKGROUND: The development of infections with ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) remains a serious problem in recipients of stem cell or organ transplants. Nearly all GCV-resistant clinical isolates have mutations in the viral UL97 gene. The rapid detection of GCV-resistant HCMV infections is necessary and the relative proportions of wild-type and mutant strains are predictive for the efficiency of antiviral therapy. To date, genotypical resistance screening has been limited to restriction fragment length polymorphism (RFLP) and sequencing analyses. Here, we present a comprehensive real-time PCR approach for the detection of most frequent mutations in the UL97 gene associated with GCV resistance. METHODS: The laboratory strains AD169 and Towne, different wild-type isolates and plasmids constructed by site-directed mutagenesis and overlap extension with specific point-mutations in the UL97 gene were analysed by LightCycler PCR and compared with UL97 RFLP and sequencing analyses. RESULTS: A new and comprehensive set of LightCycler PCRs was created using specific hybridization probes with melting-point analysis for the relevant codons 594, 595, 603 and 607. Different wild-type isolates and plasmids containing specific UL97 mutations conferring GCV resistance were investigated in the real-time PCR assay. Total processing time was 80 min per assay, whereas combinations of RFLP and sequencing needed at least 3-4 days. Proportions of co-existing wild-type and mutant strains in mixed viral populations can be obtained. CONCLUSIONS: We established a rapid real-time PCR approach for the detection of most frequent HCMV UL97 mutations associated with GCV resistance. Moreover, the method allows semiquantitative differentiation of the proportions of co-existing wild-type and mutant strains. This approach represents a new alternative for laborious RFLP analysis.

Thymidine kinase sequence analysis of herpes simplex virus type 1 strains present in different compartments in an atypical impetiginous rash on the lesional skin of a burn patient.

We report the case of a 23-year-old male burn patient with an unusual herpes simplex virus (HSV) skin manifestation. The clinical symptoms and results of HSV type 1 (HSV-1) UL23 polymorphism analysis from saliva and lesional skin underscores the need for performing molecular analysis of HSV-1 infections in burned patients presenting unusual skin lesions.

Cytomegalovirus transmission to preterm infants during lactation.

Breastfeeding has a major impact on HCMV epidemiology. The incidence of postnatal HCMV reactivation during lactation equals the maternal seroprevalence. Infectious virus, viral DNA and RNA can be isolated easily from cell and fat-free milk whey. Early onset of viral DNAlactia and virolactia as well as high viral load in milk whey are maternal risk factors for virus transmission. The dynamics of HCMV reactivation can be described by unimodal kinetics with interindividual variation. Virus reactivation during lactation is a self-limiting local process in the absence of systemic HCMV infection. Preterm infants below 1000g birthweight and a gestational age below 30 weeks may be at high risk of acquiring a symptomatic HCMV infection. Several recent studies described low transmission rates and mostly asymptomatically infected neonates using frozen milk. Despite different freeze-storing procedures, HCMV transmissions occurred, and severe HCMV infections were observed. Few data exist on the long-term outcome of postnatally acquired HCMV infection via breast milk. To substantiate the international debate on the use of native or inactivated milk for feeding of preterm infants, additional data are necessary for better identification of mother-infant-pairs at risk for viral transmission and symptomatic infection early after birth.

Effect of serum and CTL on focal growth of human cytomegalovirus.

BACKGROUND: In immunocompromised patients only cytotoxic T-lymphocytes (CTL) but not antiviral antibodies appear to be efficient in control of human cytomegalovirus (HCMV) infection. This is contrasted by the well-documented neutralising activity of patient sera against standard HCMV strains. OBJECTIVE: We tested the hypothesis that a cell-culture model based on a recent clinical HCMV isolate would more accurately approximate the clinical situation and provide an explanation for the failure of neutralising antibodies in efficient restriction of HCMV infection. METHODS: Sera from five bone marrow transplant recipients with or without prolonged HCMV replication were analysed by an enzyme-linked immunoassay for their capacity to neutralise cell-free HCMV preparations. The inhibitory effect of these sera on viral cell-to-cell-spread was then quantified by focus expansion assays using a recent clinical HCMV-isolate and was finally compared to the inhibitory effect of HCMV-specific CTL lines. RESULTS: Prolonged HCMV replication occurred in three patients despite high titres of neutralising antibodies. In contrast to the strong inhibitory effect on cell-free HCMV, their sera could not inhibit the focal growth of a recent cell-associated HCMV isolate, whereas CTL clones directed against pUL123 or pUL83 of HCMV effectively limited focal expansion of the clinical isolate in fibroblast culture. CONCLUSIONS: Focus expansion assays based on a cell-associated clinical HCMV isolate provide a model for the in vivo effectiveness of virus-specific CTL and neutralising antibodies. Our data support the assumption that due to their strict cell-association clinical HCMV strains are withdrawn from neutralising antibodies.

Human cytomegalovirus glycoprotein polymorphisms and increasing viral load in AIDS patients.

BACKGROUND: Multiple strains infection of human cytomegalovirus (HCMV) was found to be correlated with increased viral load in immunodeficient patients. However, the pathogenic mechanism underlying this correlation remains unclear. To evaluate genetic polymorphisms of HCMV glycoprotein and their potential role in its viral load, HCMV glycoprotein B, N, and O (gB, gN and gO) genotypes was studied in the population of HCMV infected acquired immune deficiency syndrome (AIDS) patients. The association between glycoprotein polymorphisms and HCMV viral load was analyzed. METHODS: The genetic polymorphisms of glycoprotein from sera of 60 HCMV infected AIDS patients was investigated by multiplex nested PCR and sequencing. HCMV viral load was evaluated by quantitative PCR. RESULTS: gB1, gO1a, and gN4a were the predominant glycoprotein genotypes in HCMV infected AIDS patients and composed 86.96%, 78.8%, and 49.2%, respectively. Only gN4a genotype infection significantly increased viral load (P = 0.048). 71% (43/60) of HCMV infected AIDS patients were found to carry multiple HCMV strains infection. A novel potential linkage of gO1a/gN4a was identified from multiple HCMV infected patients. It was the most frequent occurrence, accounted for 51.5% in 33 patients with gO and gN genotypes infection. Furthermore, the gO1a/gN4a linkage was correlated to an increased viral load (P = 0.020). CONCLUSION: The gN4a correlates to higher level HCMV load in AIDS patients. Interestingly, a novel gO1a/gN4a linkage is identified from the patients with multiple HCMV strains infection and is also associated with an increased viral load. Therefore, the pathogenic mechanism underlying glycoprotein polymorphisms and interaction of variants should be analyzed further.

Comparison of cytomegalovirus (CMV)-specific neutralization capacity of hyperimmunoglobulin (HIG) versus standard intravenous immunoglobulin (IVIG) preparations: Impact of CMV IgG normalization.

BACKGROUND: Based on a non-randomized study of Nigro et al. (2005) the intravenous administration of hyperimmunoglobulins (HIGs) is applied frequently to women with primary CMV-infection as "off-label use" in Germany. OBJECTIVES: In order to describe their CMV-specific neutralization-capacity in vitro, we analyzed the HIG preparations Cytotect®, and Cytogam® as well as the standard intravenous immunoglobulins (IVIG) Octagam®, Gamunex®, Kiovig®. STUDY DESIGN: We performed short-term cell-free CMV neutralization assays (CFNT) and long-term cell-adapted neutralization-plaque-reduction assays (PRANT). Human retinal epithelial cells (ARPE-19) were used as target cells. A clinical CMV primary-isolate from amnion fluid propagated in epithelial cells without any initial fibroblast adaption was used. For calibration we previously generated serum-pools (N=100) from two cohorts of mothers at birth: seronegative and latently CMV-infected mothers. Biochemical analysis included total protein, albumin, Ig-class, and IgG-subclasses. Additionally, CMV antibody-reactivity was checked using recombinant immunoblotting. RESULTS: HIG and IVIG preparations showed differences in levels and patterns of protein, Ig-class and CMV-specific antibody concentrations. All IgG-preparations showed high in vitro NT-capacity and high IgG-avidity. The NT90-values for HIGs and IVIGs and our seropositive reference-pool showed similar NT-capacity at a dilution of (1:100) which corresponded well to 4.1 PEI-Units/ml. CONCLUSION: All HIG- and IVIG-preparations showed similar NT-capacity following CMV IgG-normalization. Our in vitro results are in strong contrast to former findings suggesting higher functional CMV NT titers in IVIG-preparations compared to HIGs.

Presentation of a Conserved Adenoviral Epitope on HLA-C*0702 Allows Evasion of Natural Killer but Not T Cell Responses.

Infection with adenovirus is a major cause of infectious mortality in children following hematopoietic stem-cell transplantation. While adoptive transfer of epitope-specific T cells is a particularly effective therapeutic approach, there are few suitable adenoviral peptide epitopes described to date. Here, we describe the adenoviral peptide epitope FRKDVNMVL from hexon protein, and its variant FRKDVNMIL, that is restricted by human leukocyte antigen (HLA)-C*0702. Since HLA-C*0702 can be recognized by both T cells and natural killer (NK) cells, we characterized responses by both cell types. T cells specific for FRKDVNMVL were detected in peripheral blood mononuclear cells expanded from eight of ten healthy HLA-typed donors by peptide-HLA multimer staining, and could also be detected by cultured interferon γ ELISpot assays. Surprisingly, HLA-C*0702 was not downregulated during infection, in contrast to the marked downregulation of HLA-A*0201, suggesting that adenovirus cannot evade T cell responses to HLA-C*0702-restricted peptide epitopes. By contrast, NK responses were inhibited following adenoviral peptide presentation. Notably, presentation of the FRKDVNMVL peptide enhanced binding of HLA-C*0702 to the inhibitory receptor KIR2DL3 and decreased NK cytotoxic responses, suggesting that adenoviruses may use this peptide to evade NK responses. Given the immunodominance of FRKDVNMVL-specific T cell responses, apparent lack of HLA-C*0702 downregulation during infection, and the high frequency of this allotype, this peptide epitope may be particularly useful for adoptive T cell transfer therapy of adenovirus infection.