Differential kinetics of human cytomegalovirus load and antibody responses in primary infection of the immunocompetent and immunocompromised host.

The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. In total, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4(+) and CD8(+) HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, with the two antibody levels then overlapping from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting for up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titres in the immunocompetent over the entire one-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4(+) T cells. In this setting, the elevated antibody response occurred in the presence of differentiated follicular helper T cells in the blood, which decreased in number as did antibody titres upon reappearance of HCMV-specific CD4(+) T cells.

Kinetics of effector functions and phenotype of virus-specific and γδ T lymphocytes in primary human cytomegalovirus infection during pregnancy.

The T-cell response to human cytomegalovirus (HCMV) primary infection was analyzed in 27 pregnant women during the first year after primary HCMV infection. Pregnant women with remote HCMV infection were enrolled as controls. Interferon-γ-producing T cells were readily detected at levels comparable (CD4(+)) or higher (CD8(+)) than controls, whereas the CD4(+) and CD8(+) lymphoproliferative response as well as IL-2 production was significantly reduced with respect to controls for at least 9 months after infection. In addition, CD45RA re-expression as well as cytotoxic T lymphocyte activity and perforin expression were the major components of the adaptive CD4(+) and CD8(+) T-cell immune response, while Vδ2(-) γδ T-cell expansion in response to HCMV infection followed kinetics similar to that of CD8(+) T cells. Reduced CD45RA re-expression directly correlated with HCMV transmission to the fetus, thus providing an important prognostic parameter.

Human cytomegalovirus tropism for endothelial/epithelial cells: scientific background and clinical implications.

Human cytomegalovirus (HCMV) has been routinely isolated from and propagated in vitro in human embryonic lung fibroblast (HELF) cell cultures, while in vivo it is known to infect predominantly endothelial and epithelial cells. In recent years, genetic determinants of the HCMV tropism for endothelial/epithelial cells were identified in the UL131A/UL130/UL128 locus of HCMV genome of wild-type strains. UL131A-UL128 gene products form a complex with glycoprotein H (gH) and L (gL) resulting in a gH/gL/UL131A-UL128 complex that is required for HCMV entry into endothelial/epithelial cells. In contrast, virus entry into fibroblasts has its genetic determinants in the complex gH/gL/gO (or gH/gL). During primary HCMV infection, the neutralising antibody response measured in endothelial cells (EC) is potent, occurs very early and is directed mostly against combinations of two or three gene products of the UL131A-128 locus. On the contrary, neutralising antibodies measured in fibroblasts appear late, are relatively weak in potency and are directed against gH and gB. The T-cell immune response to UL131A-UL128 gene products remains to be investigated. Recently, a role has been proposed for neutralising antibody in conferring prevention/protection against HCMV infection/disease in pregnant women with primary HCMV infection. However, the level of cooperation between humoral immunity and the well-established T-cell protection remains to be defined.

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

BACKGROUND: Human cytomegalovirus infections are mostly asymptomatic in infants and young children, while they are often associated with overt clinical symptoms in adults. OBJECTIVES: To verify whether the antibody response to HCMV is more potent in symptomatic non-pregnant adults as compared to asymptomatic/paucisymptomatic pregnant women. STUDY DESIGN: Overall, 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. RESULTS: In 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. CONCLUSIONS: The 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).

Pathogenesis of human cytomegalovirus infection and cellular targets.

In acquired immunodeficiency syndrome patients with human cytomegalovirus (HCMV), disseminated infection, and end-organ disease, autopsy findings show a generalized HCMV infection of endothelial cells. On the other hand, immunocompromised transplanted patients show presence of virus and virus products in peripheral blood leukocytes (PBL), when affected by a disseminated HCMV infection. All diagnostic assays are based on the detection of virus and viral components in PBL or whole blood, including polymorphonuclear leukocytes and monocytes. The interplay between endothelial cells and leukocytes represents the pathogenetic basis for all clinical syndromes originating during disseminated HCMV infections and is the trigger for the transmission of HCMV from mother to fetus during primary infections of pregnant women. The two biologic properties of endothelial cell tropism and leukocyte (polymorphonuclear- and monocyte-) tropism are shared by all recent clinical HCMV isolates, whereas they are missing in laboratory-adapted strains. The potential role of HCMV in the pathogenesis of atherosclerosis both in the immunocompetent (after angioplasty) and the heart transplant patient is receiving support from seroepidemiologic findings, in vivo animal models, in vitro data, and also some clinical observations. The interaction of endothelial cells and leukocytes with subsequent spreading of infection to smooth muscle cells may be a major pathogenetic mechanism at the basis of this important vascular disease.

Adoptive Transfer of Herpesvirusspecific Cytotoxic T Lymphocytes in Transplant Recipients.

Profound suppression of T-cell immunity observed after transplantation may permit the emergence of life-threatening viral complications. Prevention or treatment of viral infections in immunocompromised patients through the infusion of small numbers of antigen-specific T-cell lines or clones is the most successful example of adoptive immunotherapy in humans. In fact, adoptive transfer of T cells of donor origin has been demonstrated to be able to restore protective immunity towards these infectious agents and to control established disease. This article reviews human trials of adoptive immunotherapy for herpesvirus-related complications, with the aim of defining principles and problems encountered so far to aid the design of future clinical studies.

Rubella control in Italy.

In Italy, rubella vaccination has been recommended since 1972 for pre-adolescent girls, and since the early 1990s for all children in the second year of life. Nevertheless, coverage in children from 12 to 24 months of age is suboptimal (i.e., 56% in 1998, 78% in 2003), with wide variations among regions. As a result, rubella is still circulating in Italy, and in 1996 the percentage of women susceptible to rubella between 15 and 39 years of age was >5%. Congenital rubella syndrome (CRS) was a notifiable disease between 1987 and 1991, with a range of 8-76 cases reported annually. Since 1992, national incidence data are no longer available, but local reports show that CRS cases are still occurring. Nationwide, coordinated and uniform actions are needed to control CRS effectively. For this reason, the National Plan for the Elimination of Measles and of Congenital Rubella has recently been launched. This plan includes strategies aimed at increasing MMR vaccination coverage in children and specific control measures for congenital rubella control, i.e., improving the vaccination of susceptible women of childbearing age, and reintroducing national surveillance of CRS.

Serum antibody response to the gH/gL/pUL128-131 five-protein complex of human cytomegalovirus (HCMV) in primary and reactivated HCMV infections.

BACKGROUND: Recently, a new human cytomegalovirus (HCMV) glycoprotein complex has been identified and potentially proposed as a vaccine. OBJECTIVE: The aim of this study was to determine whether the HCMV gH/gL/pUL128-pUL130-pUL131 (gH/gL/pUL128-131) 5-protein (pentameric) complex (which has been recently found to be indispensable for the infection of endothelial and epithelial cells) is able to elicit a consistent antibody response in both primary and reactivated HCMV infections. STUDY DESIGN: The antibody response was determined by both indirect immunofluorescence (IFA) and ELISA, using fixed (IFA) or lysed (ELISA) epithelial (ARPE-19) cells infected with one or more adenoviral vectors, each carrying one HCMV gene and, in parallel, with a control adenovirus vector. RESULTS: The specificity of results was determined by the reactivity of human neutralizing mAbs recognizing two, three, or four proteins of the complex. In 14 cases of primary infection, an IgG antibody seroconversion to the UL128-131 gene products was consistently detected within 2-4 weeks after onset of infection, while antibodies persisted for at least 12 months. The IgG antibody response to UL128-131 gene products was generally superior to the response to gH and appeared to follow the neutralizing antibody response (as determined in epithelial cells). In reactivated infections, the antibody response showed a trend reminiscent of a booster response. IgG antibodies were detected in HCMV-seropositive healthy adult controls, but not in HCMV-seronegative individuals. CONCLUSIONS: The IgG antibody response to the pentameric complex could be a major target for the evaluation of the antibody response to a pentamer-based vaccine.

Human cytomegalovirus serum neutralizing antibodies block virus infection of endothelial/epithelial cells, but not fibroblasts, early during primary infection.

A panel of human sera exhibited a >or=128-fold higher neutralizing potency against a human cytomegalovirus (HCMV) clinical isolate propagated and tested in endothelial (or epithelial) cells than against the same virus infecting human fibroblasts. In a group of 18 primary infections, the reverse geometric mean titre was in the range of 10-15 in human fibroblasts within the first 3 months after the onset of infection, whereas the endothelial cell infection-neutralizing activity was already present within the first 10 days, reaching median levels of 122, 320 and 545 at respectively 30, 60 and 90 days after onset, then declining slowly. This difference was also confirmed in the majority of reactivated and remote HCMV infections, as well as in a hyperimmune globulin preparation. The antibody response to HCMV pUL131A, pUL130 and pUL128 locus products, which are required for endothelial/epithelial cell infection, provided a potential molecular basis for such a differential neutralizing activity. In addition, monoclonal/monospecific antibodies raised against the pUL131A, pUL130 and pUL128 proteins were found to display an inhibitory activity on HCMV plaque formation and HCMV leukocyte transfer from HCMV-infected cells. Hence, conventional determination of the neutralizing activity of human sera in fibroblasts is misleading. Antibodies to pUL131A, pUL130 and pUL128 appear to display a major HCMV-neutralizing and dissemination-inhibiting activity.

Impact of human metapneumovirus and human cytomegalovirus versus other respiratory viruses on the lower respiratory tract infections of lung transplant recipients.

UNLABELLED: Viral respiratory tract infections in lung transplant recipients may be severe. During three consecutive winter-spring seasons, 49 symptomatic lung transplant recipients with suspected respiratory viral infection, and 26 asymptomatic patients were investigated for presence of respiratory viruses either in 56 nasopharyngeal aspirate or 72 bronchoalveolar lavage samples taken at different times after transplantation. On the whole, 1 asymptomatic (3.4%) and 28 symptomatic (57.1%) patients were positive for human metapneumovirus (hMPV, 4 patients), influenza virus A (3 patients), and B (2 patients), respiratory syncytial virus (2 patients), human coronavirus (2 patients), human parainfluenza virus (2 patients), rhinovirus (5 patients), while 4 patients were coinfected by 2 respiratory viruses, and 5 were infected sequentially by 2 or more respiratory viruses. In bronchoalveolar lavage samples, hMPV predominated by far over the other viruses, being responsible for 60% of positive specimens, whereas other viruses were present in nasopharyngeal aspirates at a comparable rate. RT-PCR (detecting 43 positive samples/128 examined) was largely superior to monoclonal antibodies (detecting 17 positive samples only). In addition, HCMV was detected in association with a respiratory virus in 4/18 HCMV-positive patients, and was found at a high concentration (>10(5) DNA copies/ml) in 3/16 (18.7%) patients with HCMV-positive bronchoalveolar lavage samples and pneumonia. Coinfections and sequential infections by HCMV and respiratory viruses were significantly more frequent in patients with acute rejection and steroid treatment. IN CONCLUSION: (i) about 50% of respiratory tract infections of lung transplant recipients were associated with one or more respiratory viruses; (ii) hMPV largely predominates in bronchoalveolar lavage of symptomatic lung transplant recipients, thus suggesting a causative role in lower respiratory tract infections; (iii) RT-PCR appears to be the method of choice for detection of respiratory viruses in lung transplant recipients, (iv) a high HCMV load in bronchoalveolar lavage is a risk factor for viral pneumonia, suggesting some measure of intervention for the control of viral infection.

Rapid detection of human metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal antibodies.

Monoclonal antibodies to human metapneumovirus (hMPV) were developed for direct fluorescent antibody (DFA) staining of nasopharyngeal aspirates from 40 infants with respiratory infections, as well as for hMPV identification in shell vial cultures. With reference to reverse transcription-PCR, DFA staining showed sensitivity, specificity, and positive and negative predictive values of 73.9%, 94.1%, 94.4%, and 72.7%, respectively. Monoclonal antibodies are useful for direct hMPV detection.

Dendritic-cell infection by human cytomegalovirus is restricted to strains carrying functional UL131-128 genes and mediates efficient viral antigen presentation to CD8+ T cells.

Human cytomegalovirus (HCMV) genetic determinants of endothelial-cell tropism and virus transfer to leukocytes (both polymorphonuclear and monocyte) have been recently identified in the UL131-128 genes. Here it is documented that the same genetic determinants of HCMV are responsible for monocyte-derived dendritic-cell (DC) tropism, i.e. all endotheliotropic and leukotropic strains of HCMV are also DC-tropic (or dendrotropic). In fact, all recent clinical HCMV isolates and deletion mutants sparing the UL131-128 locus as well as the endotheliotropic revertants AD169 and Towne were able to productively infect DC following co-culture with infected endothelial cells. On the contrary, the same clinical isolates extensively propagated in human fibroblasts, the UL131-128 deletion mutants and the reference laboratory strains were not. Peak extracellular virus titres in DC were reached 4-7 days post-infection (p.i.). Viral proteins pp65 and p72 were detected 1-3 h p.i., involving the great majority of DC 24 h p.i., while gB was abundantly detected 96 h p.i., when a cytopathic effect first appeared. Infection of DC with cell-free virus released into the medium could only be achieved with HCMV strains extensively adapted to growth in endothelial cells, reaching the peak titres 10 days p.i. DC infected for 24 h with cell-free virus and incubated for 16 h with autologous peripheral blood mononuclear cells were found to act as a potent stimulator of both HCMV-specific CD4+- and CD8+-mediated immune responses, as determined by cytokine flow cytometry. DC incubated with inactivated crude whole viral antigen preparations were only capable of eliciting a significant CD4+-mediated immune response.

Monoclonal antibodies versus reverse transcription-PCR for detection of respiratory viruses in a patient population with respiratory tract infections admitted to hospital.

UNLABELLED: In the winter season 2001-2002, 239 nasopharyngeal aspirate and 15 bronchoalveolar lavage samples from 208 patients (135 pediatric and 73 adults, including 19 lung transplant recipients) admitted to hospital because of an acute respiratory tract infection were examined for rapid diagnosis of respiratory viruses by two diagnostic approaches: immunological, using specific monoclonal antibodies (MAb); and molecular, using specific reverse transcription (RT)-PCR assays. Both methods detected influenza viruses A (H1N1 and H3N2) and B, human parainfluenza virus types 1 to 3, human respiratory syncytial virus (hRSV) types A and B, and human adenoviruses. In addition, human coronavirus (hCoV) groups I (229E-like) and II (OC43-like), as well as the new human metapneumovirus (hMPV), types A and B, were searched for by RT-PCR alone. When results obtained by both methods were added, the overall percentage of patients positive for at least one respiratory virus peaked at 44.2%, involving 92/208 patients (81 pediatric, and 11 adults), while 116 patients (55.8%) were negative for any respiratory virus tested. The most common circulating virus was hRSV, infecting 54 (25.9%) patients (24 type A, and 30 type B strains), followed by hMPV, infecting 12 (5.8%) patients (7 type A and 5 type B strains). Coinfections by two respiratory viruses interested 11 (5.3%) patients, and 9 (81.8%) of these were infected by hRSV in association with another respiratory virus. In the great majority of infected children, hRSV and hMPV were associated with lower respiratory tract infections. In lung transplant recipients, viruses present in bronchoalveolar lavage appeared to be associated frequently with lower respiratory tract infections. IN CONCLUSION: the combination of immunological and molecular assays is the most sensitive approach to the diagnosis of respiratory viral infections; and infections caused by the less investigated hCoVs and hMPVs represent a fair proportion of respiratory infections.

Human cytomegalovirus and human umbilical vein endothelial cells: restriction of primary isolation to blood samples and susceptibilities of clinical isolates from other sources to adaptation.

In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 examined. Analysis of other clinical samples inoculated as controls revealed, in the presence of highly infected HELF monolayers, either the presence of very few infected HUVEC with urine specimens (n = 10 samples) or the lack of infected HUVEC with throat washes (n = 3) or amniotic fluid samples (n = 2). Thus, peripheral blood leukocytes (PBL) appear essential for primary isolation of HCMV in HUVEC. In this respect, HCMV strains, recovered from clinical samples other than buffy coats in HELF only, could be readily adapted to growth in HUVEC by coculturing PBL from healthy blood donors with infected HELF and then inoculating infected PBL onto HUVEC. Recently elucidated mechanisms of interaction of leukocytes and HUVEC with bidirectional transfer of virus seem to provide the basis for the restriction of HCMV primary isolation in HUVEC to blood samples. However, virus strains recovered from only HELF could be adapted to growth in HUVEC when inoculated with HELF-derived (either cell-associated or cell-free) HCMV strains upon primary isolation. In conclusion, due to the in vitro selection of virus variants provided with both PBL tropism and HUVEC tropism, HCMV recovery in HUVEC is PBL mediated and substantially restricted to blood samples. Lack of HCMV recovery in HUVEC from clinical samples other than blood leads to the assumption that epithelial cells, such as urinary, amniotic, or pharyngeal cells, do not possess adequate adhesion molecules to establish close contacts with HUVEC.

The attenuated Towne strain of human cytomegalovirus may revert to both endothelial cell tropism and leuko- (neutrophil- and monocyte-) tropism in vitro.

The Towne strain of human cytomegalovirus (HCMV), originally recovered from the urine of a congenitally infected newborn, was attenuated through 125 passages in human embryonic lung fibroblast cell cultures. Although reliable markers of attenuation were not identified, the virus was shown to be attenuated by inoculation of both healthy human volunteers and immunocompromised patients. More recently, Towne (like other laboratory-adapted strains) was shown not to have two biological properties typical of recent clinical isolates: endothelial cell tropism and polymorphonuclear leukocyte tropism. These markers of attenuation are lost by all clinical isolates on extensive propagation in cell cultures and are apparently associated with one another. Here, we show that Towne may reacquire both endothelial cell tropism and leuko- (polymorphonuclear- and monocyte-) tropism on adaptation to growth in endothelial cell cultures. However, reversion to endothelial cell tropism is dissociated from reversion to leukotropism, since the latter was reacquired 10-20 passages later. Thus, these two biological properties, which were considered to be encoded by the same viral gene(s), appear to be distinct. Both restriction fragment length polymorphism and Southern blot analysis demonstrated the identity of the attenuated and endothelial cell tropic variants of Towne, thus suggesting that only minor variations (mutations) of the viral genome may be responsible for loss or reacquisition of the two biological properties. Viral genes involved in endothelial cell tropism and leukotropism remain to be identified. However, reversion of attenuated strains to pathogenicity in vivo cannot be excluded a priori.

The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics.

An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RV Delta UL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.

Prenatal diagnosis of congenital human cytomegalovirus infection in amniotic fluid by nucleic acid sequence-based amplification assay.

Nucleic acid sequence-based amplification assays for detection of human cytomegalovirus (HCMV) immediate-early and pp67 mRNA in 65 amniotic fluid samples tested for prenatal diagnosis of congenital HCMV infection showed sensitivity, specificity, and negative and positive predictive values >90%.

Rescue of human cytomegalovirus strain AD169 tropism for both leukocytes and human endothelial cells.

Endothelial cell-tropism- and leukocyte- (polymorphonuclear- and monocyte-) tropism (leukotropism) are two important biological properties shared by all recent clinical isolates of human cytomegalovirus (HCMV). These properties are lost during extensive propagation of HCMV isolates in human fibroblasts, as shown by reference laboratory-adapted strains AD169 and Towne. Here we show that strain AD169 may reacquire both properties in vitro, endothelial (both venous and arterial) cell-tropism preceding leukotropism (predominantly involving monocytes). Restriction fragment length polymorphism analysis and sequencing performed on the original virus inoculum from human fibroblasts and serial passages on endothelial cells confirmed virus identity. Thus, fundamental biological properties may be lost and reacquired in vitro according to the cell culture system employed. The lack of a 15 kb DNA fragment in the strain AD169 genome does not prevent the rescue of these biological functions, thus indicating that they are likely to be encoded by viral genes located elsewhere.

Genomes of the endothelial cell-tropic variant and the parental Toledo strain of human cytomegalovirus are highly divergent.

The low-passage Toledo strain of human cytomegalovirus (HCMV) and fresh clinical HCMV isolates have been reported to share the capacity to propagate efficiently in endothelial cell cultures. In the laboratory, however, repeated attempts to adapt the Toledo strain to growth in endothelial cells have been unsuccessful. Southern blot analysis of the entire viral genome and restriction length polymorphism analysis of multiple genome regions amplified by PCR demonstrated that the reported endothelial cell-tropic viral variant of the Toledo strain and the parental Toledo strain are highly divergent. In fact, the restriction profile of the genome of the endothelial cell-tropic variant seems highly distinct from that of the parental strain. In conclusion, the degree of dissimilarity between the two genomes suggests that the endothelial cell-tropic variant of the Toledo strain could have originated from a recombination event.