Primary HCMV Infection Research Articles

Ophthalmic Complications, Diagnosis, and Treatment of Congenital Human Cytomegalovirus Infection.

Background: Human cytomegalovirus (hCMV) is the most common etiological agent of congenital infections seen in newborns. Among the most commonly observed complications in children with congenital human cytomegalovirus infection are those affecting the visual system. Ocular complications of congenital CMV (cCMV) are a topic rarely addressed in the literature, which prompted the authors to update the available knowledge with the latest data. Methodology: English-language literature published between April 2000 and November 2023 (PubMed, NIH, Google Scholar) was analyzed for ocular complications of cCMV. The data obtained were categorized according to the ocular area involved and the incidence. A compilation of criteria for the symptomatic form of cCMV was also created. Results: The cCMV complications described in the literature affect all parts of the visual system: the anterior segment, the posterior segment, the posterior visual pathways, and the visual cortex. The most commonly described ocular complication of cCMV is choroidal and retinal scarring. Conclusions: Ophthalmic complications of cCMV can cause severe visual disturbances. Ophthalmic diagnosis in newborns should include hCMV PCR testing, which has the highest sensitivity and specificity. In the symptomatic form of cCMV, treatment should be instituted according to recommendations. A consensus should be established for screening of primary hCMV infection in pregnant women, the way in which to define the symptomatic form of cCMV, and the appropriateness and standards of treatment for primary hCMV infection in pregnant women.

Human cytomegalovirus preferentially infects early gestation placental macrophages and evades their antiviral immunogenicity through evasion of the type I IFN response

Abstract Human cytomegalovirus (HCMV) is the leading cause of congenital viral infections. When a pregnant woman develops primary HCMV infection in early gestation, the risk of transplacental infection is ~40%. Studies have documented the presence of HCMV in placental macrophages (Hofbauer cells [HCs]) as a consequence of maternal viremia. However, the interplay between HCMV and placental macrophages (HCs) is poorly defined. With written informed consent, placentae were collected from 12 HIV-1/Hep B/HCMV seronegative women (>18 years). HCs were exposed to human HCMV (TB40/E). qPCR, FACS, ELISA, and Western blot analysis determined the expression of activation markers, cytokines, antiviral genes, and host proteins. Here we found that HCs isolated from early gestation (15–23 weeks) placentae are more susceptible to HCMV infection than HCs isolated from term (>39 weeks) placentae. HCMV infection of early gestation HCs led to a significant upregulation of activation markers, along with elevated secretion of proinflammatory cytokines, compared to HCMV-infected term HCs. In early gestation and term HC, HCMV rapidly induces the type I IFN pathway, leading to secretion of IFNs and strong induction of ISGs. However, HCMV diminishes type I IFN-mediated phosphorylation of STAT2, with early gestation HCs exhibiting the greatest level of inhibition. Our findings show that HCMV can subvert type I IFN signaling to support viral growth at the placenta, particularly during early gestation. Studying mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections.

2651. Protection Against Human Cytomegalovirus Acquisition Is Associated with IgG Binding to Cell-Associated CMV glycoprotein B in Two Historical gB/MF59 Vaccine Cohorts

BackgroundHuman cytomegalovirus (CMV) is the most common congenital infection worldwide. A CMV glycoprotein B (gB) subunit vaccine with MF59 adjuvant achieved ~50% protection in phase II clinical trials in postpartum and adolescent women. Interestingly, postpartum vaccinees showed poor virus neutralization but robust antibody-dependent cellular phagocytosis (ADCP). In this study, we performed a combined humoral immune correlate of risk analysis in vaccinees to define vaccine-elicited immune responses associated with protection and targets for vaccine candidate immunogenicity.MethodsgB/MF59 vaccinees who became infected and those who remained uninfected were 2:1 matched on race and number of vaccine doses. This study included 42 women from the adolescent (14 infected, 28 uninfected) and 33 from the postpartum cohorts (11 infected, 22 uninfected). IgG binding to whole gB, gB-neutralizing epitopes, FCRs, and whole virions were assessed by standard or multiplex ELISA. IgG binding to gB mRNA-transfected HEK293Ts was measured by flow cytometry. Neutralization of Towne, TB40/E, and AD169-repaired-GFP strains were measured in MRC-5, BJ5Ta, and/or ARPE-19 cells. Phagocytosis was assessed by THP-1 uptake of fluorescently conjugated TB40/E and AD169-repaired-GFP virions. Multiple linear regression controlling for cohort was performed for the combined log-transformed group data (apriori significance cut-off of P < 0.05, Benjamin–Hochberg FDR < 0.2).ResultsVaccine-elicited antibodies in adolescent and postpartum cohorts exhibited similar magnitude IgG binding to soluble HCMV gB protein, gB-neutralizing domains, and gB-transfected cells. Autologous Towne strain neutralization was observed in both cohorts, but heterologous strain neutralization was observed only in adolescent vaccinees (P = 0.001). Both cohorts exhibited robust phagocytosis of HCMV virions. Regression analyses revealed that risk of HCMV acquisition in vaccinees was associated with magnitude IgG binding to gB-transfected cells (P = 0.006, FDR = 0.15), not neutralization or phagocytosis responses.ConclusionProtection against primary HCMV infection was significantly associated with vaccine-elicited IgG binding to gB-transfected cells, suggesting the importance of a native, cell-associated gB conformation in future vaccine candidates.Disclosures All authors: No reported disclosures.

Expression of IL-7Rα and KLRG1 defines functionally distinct CD8+ T-cell populations in humans.

During acute viral infections in mice, IL‐7Rα and KLRG1 together are used to distinguish the short‐lived effector cells (SLEC; IL‐7RαloKLRGhi) from the precursors of persisting memory cells (MPEC; IL‐7RαhiKLRG1lo). We here show that these markers can be used to define distinct subsets in the circulation and lymph nodes during the acute phase and in “steady state” in humans. In contrast to the T cells in the circulation, T cells derived from lymph nodes hardly contain any KLRG1‐expressing cells. The four populations defined by IL‐7Rα and KLRG1 differ markedly in transcription factor, granzyme and chemokine receptor expression. When studying renal transplant recipients experiencing a primary hCMV and EBV infection, we also found that after viral control, during latency, Ki‐67‐negative SLEC can be found in the peripheral blood in considerable numbers. Thus, combined analyses of IL‐7Rα and KLRG1 expression on human herpes virus‐specific CD8+ T cells can be used to separate functionally distinct subsets in humans. As a noncycling IL‐7RαloKLRG1hi population is abundant in healthy humans, we conclude that this combination of markers not only defines short‐lived effector cells during the acute response but also stable effector cells that are formed and remain present during latent herpes infections.

The Transcription Factor Hobit Identifies Human Cytotoxic CD4+ T Cells.

The T cell lineage is commonly divided into CD4-expressing helper T cells that polarize immune responses through cytokine secretion and CD8-expressing cytotoxic T cells that eliminate infected target cells by virtue of the release of cytotoxic molecules. Recently, a population of CD4+ T cells that conforms to the phenotype of cytotoxic CD8+ T cells has received increased recognition. These cytotoxic CD4+ T cells display constitutive expression of granzyme B and perforin at the protein level and mediate HLA class II-dependent killing of target cells. In humans, this cytotoxic profile is found within the human cytomegalovirus (hCMV)-specific, but not within the influenza- or Epstein–Barr virus-specific CD4+ T cell populations, suggesting that, in particular, hCMV infection induces the formation of cytotoxic CD4+ T cells. We have previously described that the transcription factor Homolog of Blimp-1 in T cells (Hobit) is specifically upregulated in CD45RA+ effector CD8+ T cells that arise after hCMV infection. Here, we describe the expression pattern of Hobit in human CD4+ T cells. We found Hobit expression in cytotoxic CD4+ T cells and accumulation of Hobit+ CD4+ T cells after primary hCMV infection. The Hobit+ CD4+ T cells displayed highly overlapping characteristics with Hobit+ CD8+ T cells, including the expression of cytotoxic molecules, T-bet, and CX3CR1. Interestingly, γδ+ T cells that arise after hCMV infection also upregulate Hobit expression and display a similar effector phenotype as cytotoxic CD4+ and CD8+ T cells. These findings suggest a shared differentiation pathway in CD4+, CD8+, and γδ+ T cells that may involve Hobit-driven acquisition of long-lived cytotoxic effector function.

Monitoring of human cytomegalovirus DNAemia during primary infection in transmitter and non-transmitter mothers

Background and objectivesIt has been reported that maternal DNAemia is detectable in three quarters of pregnant women with acute/recent primary HCMV infections, with a higher median number of HCMV DNA copies/ml blood in transmitter as compared with non-transmitter mothers. Study designThe kinetics of HCMV DNA in blood of transmitter vs non-transmitter pregnant women with primary HCMV infection was retrospectively analyzed from their first blood sampling at referral up to amniocentesis strictly performed at 19–21 weeks’ gestation. Monthly monitoring of maternal HCMV DNAemia was performed up to prenatal diagnosis. ResultsHCMV DNAemia was determined in 154 pregnant women. At amniocentesis, HCMV DNA in blood was positive in 42/50 (84.0%) amniotic fluid (AF) −positive and 21/104 (20.2%) AF-negative mothers (p<0.0001). The number of HCMV DNA copies/ml blood was not significantly different in AF-positive as compared with AF-negative mothers in the interval 0–30days post-infection (p=0.14). On the contrary, HCMV DNA load at 30–60days (p=0.03) and at 60–90days (p<0.001) after onset of infection was significantly different, as observed at amniocentesis (p<0.001). Three patterns (clearance, delayed decrease, and increasing) in both transmitter and non-transmitter mothers were observed. However, 79.8% AF- negative mothers cleared HCMV DNA in blood, while in AF-positive mothers increasing (44.0%) or persisting (40.0%) levels of DNAemia were observed. ConclusionsThe presence of viral DNA in maternal blood at amniocentesis is statistically associated with fetal HCMV infection. Increasing or persisting levels of maternal DNAemia during primary HCMV infection in pregnancy correlate with HCMV transmission to the fetus.

Primary HCMV infection in pregnancy from classic data towards metabolomics: An exploratory analysis

BackgroundHuman cytomegalovirus (HCMV) is one of the most frequent risk of viral infections during pregnancy. The aim of this study was to evaluate the metabolic profile in amniotic fluid (AF) samples obtained from HCMV-infected, and uninfected fetuses in order to elucidate changes in metabolic pathways during congenital HCMV infection and to recognize new potential diagnostic and/or prognostic biomarkers. MethodsA retrospective cohort study was conducted on 63 pregnant women: 20 contracted primary HCMV infection during pregnancy and, subsequently, transmitted the virus to the fetus (transmitters); 20 contracted the infection without transmitting the virus to the fetus (non-transmitters); 23 who underwent amniocentesis for cytogenetic-based diagnosis were considered controls. Metabolomics analysis was performed by using the hyphenated technique Gas chromatography-mass spectrometry (GC–MS) followed by a multivariate statistical approach. Four PLS-DA models were generated: controls vs. transmitters; controls vs. non-transmitters; transmitters vs. non-transmitters; and asymptomatic infected vs. symptomatic infected newborns. Subsequently, these models were exploited for network mapping. ResultsCompared with controls, HCMV transmitters showed significantly increased levels in glutamine, glycine, serine, pyruvic acid, threonine, threonic acid, and cystine; conversely, unknown U1715 and U1804, glutamic acid, U1437, fructose, sugar-like A203003 and A203005, and tyrosine levels were found decreased. In non-transmitters, glutamine, serine, glycine, threonic acid, threonine, 1-monostearin, urea, and cystine were found increased, while sorbitol, unknown U1804, sugar-like A203003, U1751, xylitol, leucine and fructose were decreased. The comparison between transmitters and non-transmitters did not produce a statistically significant model. Unlike controls' profile, a common feature of HCMV infected subjects (transmitters and non-transmitters) was the activation of glutamine-glutamate and pyrimidine metabolic pathways. In addition, a clusterization for asymptomatic vs. symptomatic outcome was also observed due to alteration of fatty acids biosynthesis. ConclusionsMetabolomics approach could highlight the significant modification of maternal and placental status during HCMV infection for both transmitter and non-transmitter subjects. A further separation was observed for asymptomatic vs. symptomatic HCMV congenital infections model. Therefore, metabolomics may be a promising tool to improve the accuracy of an early diagnosis, and the management of HCMV pregnancy-related infections.

Human Cytomegalovirus Induces an Atypical Activation of Akt To Stimulate the Survival of Short-Lived Monocytes.

Human cytomegalovirus (HCMV) is a pervasive herpesvirus responsible for significant morbidity and mortality among immunodeficient/naive hosts. Following a primary HCMV infection, circulating blood monocytes mediate the systemic spread of the virus. Extending the short 48-h life span of monocytes is critical to the viral dissemination process, as these blood-borne cells are nonpermissive for virus replication until they are fully differentiated into macrophages. Here, we show that HCMV glycoprotein gB binding to cellular epidermal growth factor receptor (EGFR) during HCMV entry initiated a rapid (within 15 min) activation of the apoptosis suppressor Akt, which was maintained through 72 h. The virus-induced activation of Akt was more robust than that with the normal myeloid growth factor macrophage colony-stimulating factor (M-CSF) and was essential for infected monocytes to bypass the 48-h viability checkpoint. Activation of phosphoinositide 3-kinase (PI3K) following EGFR engagement by HCMV mediated the phosphorylation of Akt. Moreover, HCMV entry drove a switch away from the PI3K p110δ isoform, which was required for the viability of uninfected monocytes, to the p110β isoform in order to facilitate the Akt-dependent prosurvival state within infected cells. Simultaneously, in contrast to M-CSF, HCMV promoted a rapid increase in SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) expression, leading to signaling through a noncanonical Akt activation pathway. To ensure maximum Akt activity, HCMV also induced an early phosphorylation-dependent inactivation of the negative regulator phosphatase and tensin homolog. Overall, our data indicate that HCMV hijacks the upstream Akt signaling network to induce a nontraditional activation of Akt and subsequently a prosurvival decision at the 48-h cell fate checkpoint, a vital step for HCMV's dissemination and persistence strategy. HCMV is found throughout the world with a prevalence of 55 to 100% within the human population. HCMV infection is generally asymptomatic in immunocompetent or naive individuals but is a significant cause of morbidity and mortality among the immunocompromised. Widespread organ inflammation is associated with symptomatic infections, which is a direct consequence of the viral dissemination strategy. Inflammatory peripheral blood monocytes facilitate the spread of HCMV. However, HCMV must subvert the naturally short life span of monocytes. In this work, we demonstrate that HCMV induces the activation of Akt, an antiapoptotic protein, in a manner distinct from that of normal myeloid growth factors. Moreover, we decipher how HCMV dysregulates the upstream Akt signaling network during viral entry to promote an Akt-dependent prosurvival state following infection. Delineation of the virus-specific mechanisms that regulate cellular prosurvival pathways in order to drive the survival of HCMV-infected monocytes is important to identifying new anti-HCMV therapeutic targets.

Seroprevalence and Risk of Primary Maternal HCMV Infection among Pregnant Women in Taiz City, Yemen

Seroprevalence and Risk of Primary Maternal HCMV Infection among Pregnant Women in Taiz City, Yemen

Risk of congenital disease in 46 infected fetuses according to gestational age of primary human cytomegalovirus infection in the mother.

Given the difficulty in establishing the exact time of HCMV transmission from mother to fetus, HCMV intrauterine infection was investigated in 46 infected fetuses/newborns by correlating maternal and fetal parameters with clinical outcome according to the time interval between the onset of maternal infection and prenatal diagnosis. In detail, 17/28 (60.7%) asymptomatic and 18/18 (100%) symptomatic fetuses/newborns were infected as a consequence of a primary maternal HCMV infection acquired ≤8 weeks of gestational age, while 11/28 (39.3%) asymptomatic and 0/18 (0%) symptomatic fetuses/newborns were congenitally infected when maternal infection was acquired >8 weeks' gestation. Symptomatic fetal infections appeared to be associated with a maternal primary infection occurring at ≤ 8 weeks' gestation. Cordocentesis performed at 20 weeks' gestation should be restricted to high risk infected fetuses.

Exploiting viral natural history for vaccine development.

The partial successes of the Phase 2 gB-based vaccine trials for HCMV highlight the very real likelihood that vaccine-mediated induction of antibodies that neutralize the fusion pathway of fibroblast infection is not sufficient as a singular strategy to confer protective efficacy against primary HCMV infection. Alternative strategies that serve as adjuncts to gB-based vaccines are likely required to target different aspects of the complex lifecycle of HCMV infection. There has been considerable recent interest in targeting the gH/gL/UL128/UL130/UL131 pentamer complex (gH/gL-PC) to neutralize the endocytic pathway of HCMV infection of epithelial and endothelial cells. Since both cell types are critical during primary mucosal infection, intrahost spread, and shedding of HCMV in an infected host, the gH/gL-PC represents a high-value target for vaccination to interrupt the HCMV lifecycle. The natural history of HCMV is exceedingly complex and incompletely resolved, and the protective efficacy generated by gH/gL-PC remains to be validated in clinical trials. Yet, there are salient aspects of its lifecycle that offer clues about how other novel vaccine strategies can be targeted to especially susceptible parts of the viral proteome to significantly disrupt HCMV’s ability to infect susceptible hosts. In particular, the protracted evolution of Herpesvirales has endowed HCMV with two remarkable properties of its natural history: (1) lifelong persistence within immune hosts that develop extraordinarily large antiviral immune responses and (2) the ability to reinfect those with prior immunity. The latter phenotype strongly implies that, if HCMV can overcome prior immunity to initiate a new infection, it is likely irrelevant whether prior immunity derives from prior infection or prior vaccination. Both phenotypes are unified by the extensive devotion of the HCMV coding repertoire (~50 %) to viral proteins that modulate host cell signaling, trafficking, activation, antigen presentation, and resistance to apoptosis. Collectively, these viral proteins are the likely reason for the high barrier to success for the 4-decade effort to design an HCMV vaccine, and they represent the viral proteins that make HCMV be the virus that it is. James Hanshaw wrote in 1971 that, based on a 15-year retrospective of congenital HCMV cases, “… any thoughtful program designed at prevention or treatment deserves consideration”. Drawing upon natural history data from the nonhuman primate model of HCMV persistence and pathogenesis, a “thoughtful program” is put forth that HCMV immune-modulating proteins should be considered as vaccine candidates.

HCMV seroprevalence in couples under infertility treatment

Congenital HCMV infection affects about 0.5-0.9 % of newborns in Europe. Primary prevention could effectively be implemented in screening programmes for couples treated for infertility. By supplying epidemiological data, the proportion of women susceptible to primary HCMV infection is estimated. Retrospective evaluation of HCMV IgG of couples with recurrent implantation failure in German IVF programmes referred to a tertiary immunological centre for lymphocyte immunotherapy in 2002 (727 couples) and 2012 (294 male partners only). HCMV seroprevalence was 45 % in women and 33 % in men of childbearing age in 2002 and has not declined any further in the male group (34 %) in 2012. It was higher in women (39.8 %) than in males (28.0 %) of German descent (p < 0.00001). Most couples were concordantly seropositive (20.5 %) or negative (41.8 %). Discordant partnerships comprised of seropositive women with negative partners (24.6 %) and seronegative women with positive partners (13.1 %). More than 50 % of the women are HCMV seronegative and may benefit from primary prevention. About one fourth of them live with a seropositive partner. Risks to contract HCMV via the sexual route in a long-term discordant partnership may be very low, and the advice of protective safer sex methods in this group of prospective parents is critical since it leads to a conflict of interests.

Primary human cytomegalovirus infections: Kinetics of ELISA-IgG and neutralizing antibody in pauci/asymptomatic pregnant women vs symptomatic non-pregnant subjects

BackgroundHuman cytomegalovirus infections are mostly asymptomatic in infants and young children, while they are often associated with overt clinical symptoms in adults. ObjectivesTo verify whether the antibody response to HCMV is more potent in symptomatic non-pregnant adults as compared to asymptomatic/paucisymptomatic pregnant women. Study designOverall, 36 consecutive pregnant women with primary HCMV infection were compared with 10 consecutive symptomatic non-pregnant subjects with primary HCMV infection and overt clinical symptoms. Levels of IgG antibody responses to HCMV-infected cell lysate and the pentamer gH/gL/pUL128L, gH/gL and gB HCMV glycoprotein complexes as well as neutralizing antibodies preventing infection of epithelial cells (ARPE-19) and human embryonic lung fibroblast (HELF) cells were compared at intervals of 1–30, 31–60, 61–90, 91–180 and 181–360 days after onset of infection. In parallel, viral load was quantified by real-time PCR. ResultsIn symptomatic non-pregnant subjects, the IgG responses to HCMV lysate as well as to gH/gL and ARPE-19 neutralizing antibodies were significantly higher from 31 to 60 through 180 days after infection onset. In the same patients, the IgG antibody responses to the pentamer and HELF-neutralizing antibody were significantly higher starting 90 days post-infection. ConclusionsThe presence of overt clinical symptoms is associated with a significantly higher antibody response (concomitantly with a higher viral load) in non-pregnant subjects with symptomatic primary HCMV infection as compared to pregnant women with paucisymptomatic/ asymptomatic primary infection (and lower viral load).

Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies.

The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by "self-cleaving" 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.

Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization, and function.

Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8(+) T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα(+) hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα(+) and IL-7Rα(-) subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8(+) T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8(+) T cell pool during latency or upon antigen recall. IL-7Rα(+) PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8(+) effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8(+) T cell pool. Insight into the self-renewal properties of long-lived memory CD8(+) T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8(+) T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8(+) T cell pool.

PO-0213 Impact Of Human Cytomegalovirus (hcmv) Infection On Nk Cell Receptor (nkr) Distribution In Premature Infants

Background and aims HCMV reshapes NKR distribution, promoting an expansion of CD94/NKG2C+ NK and T cells. The dynamic of this process following primary HCMV infection is ill-defined. The role of NK cells in antiviral defence in preterm infants is unknown. Aim To assess the dynamics of NKR expression in preterm infants with postnatal HCMV infection. Methods NKR expression was analysed by flow cytometry at enrolment, 4 and 10 months in 19 initially HCMV-free infants (mean GA 34 w, range 32–36 w) and 6 infants with symptomatic postnatal HCMV infection (mean GA 29 w, range 26–41 w). Results As compared to infants not infected at enrolment, those with symptomatic HCMV infection had increased proportions of NKG2C+ (40% vs 16%, p = 0.008) and KIR+ (60% vs 36%, p + T cells were also associated to HCMV infection (p Four of the 7 initially HCMV-negative infants undergoing follow-up became asymptomatically HCMV-infected by 4 months and showed significant differences in the proportions of NKG2C+ and NKG2A+ NK cells in the second and third analyses with respect to patients remaining uninfected (p NKG2C+ and KIR+ NK cell expansion in symptomatic infants persisted in the second and third samples (n = 5) and it was comparable to that observed in asymptomatic infection. Conclusions HCMV infection is associated with a specific and persistent expansion of NKG2C+ NK cells. Innate immune response plays a role in HCMV infection control in preterm infants.

Detection of human cytomegalovirus DNA in various blood components after liver transplantation

The quantification of human cytomegalovirus (HCMV DNA) by real-time PCR is currently a primary option for laboratory diagnosis of HCMV infection. However, the optimal sample material remains controversial due to the use of different PCR assays. To explore the best blood component for HCMV DNA surveillance after liver transplantation, whole blood (WB), serum (SE), and plasma (PL) specimens were collected simultaneously from targeted patients and examined for HCMV DNA using one commercially available assay. The HCMV DNA-positive rate with WB (16.67%) was higher than that with either SE or PL (8.33%, both P<0.01). Quantitative DNA levels in WB were of greater magnitude than those in SE (WB-SE mean log-transformed difference, 0.99; 95%CI=0.74-1.25; P<0.0001) and PL (WB-PL mean log-transformed difference, 1.37; 95%CI=1.07-1.66; P<0.0001). Dynamic monitoring revealed that HCMV DNA in WB was positive sooner and had higher values for a longer period of time during therapy. With earlier positive detection, higher sensitivity, and yield of greater viral loads, WB compared favorably to SE or PL and hence is recommended as the superior material for HCMV DNA surveillance after liver transplantation. In addition, infant recipients require more intensive monitoring and prophylactic care because of their higher susceptibility to primary HCMV infection.

Human cytomegalovirus induction of a unique signalsome during viral entry into monocytes mediates distinct functional changes: a strategy for viral dissemination

HCMV pathogenesis is a direct consequence of the hematogenous dissemination of the virus to multiple host organ sites. The presence of infected monocytes in the peripheral blood and organs of individuals exhibiting primary HCMV infection have long suggested that these blood sentinels are responsible for mediating viral spread. Despite monocytes being "at the right place at the right time", their short lifespan and the lack of productive viral infection in these cells complicate this scenario of a monocyte-driven approach to viral dissemination by HCMV. However, our laboratory has provided evidence that HCMV infection is able to induce a highly controlled polarization of monocytes toward a unique and long-lived proinflammatory macrophage, which we have demonstrated to be permissive for viral replication. These observations suggest that HCMV has evolved as a distinct mechanism to induce select proinflammatory characteristics that provide infected monocytes with the necessary tools to mediate viral spread following a primary infection. In the absence of viral gene products during the early stages of infection, the process by which HCMV "tunes" the inflammatory response in infected monocytes to promote viral spread and subsequently, viral persistence remains unclear. In this current review, we focus on the viral entry process of HCMV and the potential role of receptor-ligand interactions in modulating monocyte biology. Specifically, we examine the signaling pathways initiated by the distinct combination of cellular receptors simultaneously engaged and activated by HCMV during viral entry and how the acquisition of this distinct signalsome results in a nontraditional activation of monocytes leading to the induction of the unique, functional attributes observed in monocytes following HCMV infection.

Paracrine inhibition of GM-CSF signaling by human cytomegalovirus in monocytes differentiating to dendritic cells

AbstractA primary HCMV infection or virus reactivation may cause severe disease in hosts with a deficient immune system. The virus can disturb both innate and adaptive immunity by targeting dendritic cell (DC) functions. Monocytes, the precursors of DCs in vivo (MoDCs), are the primary targets of HCMV; they can also harbor latent virus. The DCs generated from infected monocytes (CMV-MoDCs) have an altered phenotype and functional defects. We have shown that CMV-MoDCs do not secrete IL-12 in response to lipopolysaccharide stimulation, cannot ingest dead cells, induce TH1 differentiation, or the proliferation of naive allogeneic CD4+ T cells. We found that the GM-CSF signaling in an entire population of CMV-MoDCs was impaired, although only half of the cells were productively infected, and that IL-6 secretion and suppressors of cytokine signaling 3 induction contributed to this bystander effect. We also showed that MoDCs derived ex vivo from monocytes of viremic patients had the same altered phenotype as CMV-MoDCs, including decreased STAT5 phosphorylation, indicating defective GM-CSF signaling. We have thus described a new mechanism of HCMV-induced immunosupression, indicated how infection may disturb both GM-CSF–dependent physiologic processes and proposed GM-CSF–based therapeutic approaches.

Prognostic markers of symptomatic congenital human cytomegalovirus infection in fetal blood

To identify fetal cord blood prognostic markers of symptomatic congenital human cytomegalovirus infection (HCMV). Retrospective observational study. Fetal medicine unit in Milan and Medical virology unit in Pavia, Italy. HCMV-infected and -uninfected fetuses of mothers with primary HCMV infection during the period 1995-2009. Overall, 94 blood samples from as many fetuses of 93 pregnant women experiencing primary HCMV infection were examined for multiple immunological, haematological and biochemical markers as well as virological markers. Congenital HCMV infection was diagnosed by detection of virus in amniotic fluid, and symptomatic/asymptomatic infections were determined by ultrasound scans, nuclear magnetic resonance imaging, histopathology or clinical examination at birth. Blood sample markers were retrospectively compared in symptomatic and asymptomatic fetuses with congenital infection. A statistical analysis was performed to determine the value of each parameter in predicting outcome. Univariate analysis showed that most nonviral and viral markers were significantly different in symptomatic (n = 16) compared with asymptomatic (n = 31) fetuses. Receiver operator characteristics analysis indicated that, with reference to an established cutoff for each marker, the best nonviral factors for differentiation of symptomatic from asymptomatic congenital infection were β(2) -microglobulin and platelet count, and the best virological markers were immunoglobulin M antibody and DNAaemia. β(2) -Microglobulin alone or the combination of these four markers reached the optimal diagnostic efficacy. The determination of multiple markers in fetal blood, following virus detection in amniotic fluid samples, is predictive of perinatal outcome in fetuses with HCMV infection.