Maria Grazia Revello

Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant

SUMMARY Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy.

Human Cytomegalovirus UL131-128 Genes Are Indispensable for Virus Growth in Endothelial Cells and Virus Transfer to Leukocytes

ABSTRACT Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium- leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

ABSTRACT Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC90] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.

Human Cytomegalovirus in Blood of Immunocompetent Persons during Primary Infection: Prognostic Implications for Pregnancy

Human cytomegalovirus (HCMV) was investigated in peripheral blood leukocytes (PBL) of 52 immunocompetent patients (40 pregnant women) with primary HCMV infection by quantitative determination of pp65 antigenemia, viremia, and leukoDNAemia. pp65 antigenemia was detected in 12 (57.1%) of 21, 4 (25%) of 16, and 0 of 10 patients examined 1, 2, and 3 months after onset, respectively. Viremia was detected in 5 (26.3%) of 19 patients during the first month only. LeukoDNAemia was detected in 20 of 20, 17 (89.5%) of 19, and 9 (47.3%) of 19 patients tested 1, 2, and 3 months after onset, respectively. Four (26.6%) of 15 patients were still DNAemia-positive at 4-6 months, whereas none were positive at >6 months. HCMV was not detected in PBL of 20 HCMV-immune donors or of 9 seropositive subjects with recurrent infection. Virus levels were low by all assays and did not correlate with clinical course of infection, intrauterine transmission, or severity of outcome. Invasive procedures in the presence of maternal leukoDNAemia did not seem to interfere with vertical transmission of HCMV infection.

Human Cytomegalovirus Replicates Abortively in Polymorphonuclear Leukocytes after Transfer from Infected Endothelial Cells via Transient Microfusion Events

ABSTRACT Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.

Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement.

Giant cells fully permissive for human cytomegalovirus (HCMV) were found to circulate, at a variable proportion, in peripheral blood of 21 out of 25 immunocompromised patients with disseminated HCMV infection. Circulating endothelial giant cells (EGC) were identified by a specific monoclonal antibody of endothelial origin and shown to express immediate-early, early, and late viral proteins. Immunostaining patterns of different viral proteins were comparable to those detected in vitro in cultured human umbilical vein endothelial cells. EGC counts > 10 were associated with high levels (> 100) of HCMV viremia and antigenemia, as well as with an overt clinical syndrome in transplanted patients, and to an untreated long lasting organ localization in AIDS patients. On the other hand, EGC counts were < 10 during disseminated HCMV infections of both transplant recipients with no apparent organ syndrome and AIDS patients with recent organ involvement. In tissue sections from AIDS patients, infected endothelial cells were found to progressively enlarge till detaching from the small vessel wall and entering blood stream. HCMV-infected EGC represent a new systemic parameter suitable for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infections.

Diagnostic and prognostic value of human cytomegalovirus load and IgM antibody in blood of congenitally infected newborns

BACKGROUND Diagnosis of congenital human cytomegalovirus (HCMV) infection relies on virus isolation from urine collected in the first 3 weeks of life. However, very little is known about the presence, levels and duration of HCMV pp65 antigenemia, viremia and DNAemia in congenitally infected newborns. OBJECTIVES To investigate the diagnostic and prognostic value of HCMV load determination in blood of newborns/infants with congenital HCMV infection. STUDY DESIGN HCMV pp65 antigenemia, viremia and DNAemia were investigated in 116 sequential peripheral blood leukocytes (PBL) samples from 41 newborns/infants with congenital HCMV infection and in 34 PBL samples from 34 uninfected newborn. Virus-specific IgM were determined in parallel on 145 sequential serum samples. RESULTS Compared to virus isolation from urine, sensitivities of DNAemia, antigenemia, viremia, and IgM determination were 100, 42.5, 28.2, and 70.7%, respectively. Specificity was 100% for all assays. Antigenemia, viremia and DNAemia levels were significantly higher and persisted longer in newborns with symptomatic infection compared to subclinically infected babies, whereas no difference was observed for virus-specific IgM antibody between the two groups. CONCLUSIONS (i) determination of viral DNA in blood at birth appears to be a sensitive and specific marker for diagnosis of congenital HCMV infection; (ii) significantly higher levels of HCMV load were detected in infants with symptomatic HCMV infection; and (iii) virus clearance from blood occurs spontaneously both in symptomatic and subclinically infected infants. However, the process takes longer in infants presenting with symptoms at birth.

Fetal Human Cytomegalovirus Transmission Correlates with Delayed Maternal Antibodies to gH/gL/pUL128-130-131 Complex during Primary Infection

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell-to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity.

Human Cytomegalovirus Proteins pp65 and Immediate Early Protein 1 Are Common Targets for CD8+ T Cell Responses in Children with Congenital or Postnatal Human Cytomegalovirus Infection

Recombinant modified vaccinia Ankara- and peptide-based IFN-γ ELISPOT assays were used to detect and measure human CMV (HCMV)-specific CD8+ T cell responses to the pp65 (UL83) and immediate early protein 1 (IE1; UL123) gene products in 16 HCMV-infected infants and children. Age at study ranged from birth to 2 years. HCMV-specific CD8+ T cells were detected in 14 (88%) of 16 children at frequencies ranging from 60 to >2000 spots/million PBMC. Responses were detected as early as 1 day of age in infants with documented congenital infection. Nine children responded to both pp65 and IE1, whereas responses to pp65 or IE1 alone were detected in three and two children, respectively. Regardless of the specificity of initial responses, IE1-specific responses predominated by 1 year of age. Changes in HCMV epitopes targeted by the CD8+ T cell responses were observed over time; epitopes commonly recognized by HLA-A2+ adults with latent HCMV infection did not fully account for responses detected in early childhood. Finally, the detection of HCMV-specific CD8+ T cell responses was temporally associated with a decrease in peripheral blood HCMV load. Taken altogether, these data demonstrate that the fetus and young infant can generate virus-specific CD8+ T cell responses. Changes observed in the protein and epitope-specificity of HCMV-specific CD8+ T cells over time are consistent with those observed after other primary viral infections. The temporal association between the detection of HCMV-specific CD8+ T cell responses and the reduction in blood HCMV load supports the importance of CD8+ T cells in controlling primary HCMV viremia.

Diagnosis and Outcome of Preconceptional and Periconceptional Primary Human Cytomegalovirus Infections

Primary human cytomegalovirus (HCMV) infection occurring in pregnant women within 3 months before (preconceptional) or within 4 weeks after (periconceptional) the last menstrual period represents an as-yet-undefined risk to the fetus. One (9.1%) of 11 newborns born to 12 women with preconceptional infection was subclinically infected (1 aborted fetus was not examined for infection). Of 20 pregnancies in women with periconceptional infection, 7 were terminated before 12 weeks of gestation (aborted fetus was not examined), 1 was terminated at 23 weeks after prenatal diagnosis of congenital infection, and 12 continued to term. Of those 12, 3 resulted in newborns who were congenitally infected. Thus, in the periconceptional group, intrauterine transmission occurred in 4 (30.8%) of 13 pregnancies for which the virologic outcome was known. One newborn was symptomatic at birth, and disseminated HCMV infection was diagnosed in an aborted fetus. Periconceptional primary HCMV infection seems to bear a higher risk of unfavorable outcome than preconceptional infection, and counseling should be adjusted accordingly.