Antibodies to the major linear neutralizing domains of cytomegalovirus glycoprotein B among natural seropositives and CMV subunit vaccine recipients.

The gB protein (gpUL55) of human cytomegalovirus (CMV) contains C-terminal (AD-1) and N-terminal (AD-2) linear immunodominant neutralizing domains. To measure antibodies to these epitopes, a modified protein (delta-gB) lacking heavily glycosylated intervening regions, the transmembrane domain, and the cytoplasmic domain, was expressed in recombinant baculovirus-infected cells. Eighty-six percent of 600 naturally CMV-seropositive individuals and 93% of 121 gB vaccine recipients had antibodies to delta-gB as detected by enzyme-linked immunosorbent assay (ELISA). The antibody level in vaccinees (median optical density [OD] = 1.73) exceeded that in natural seropositives (median OD = 0.94; p < .0001). Eleven percent of 95 natural seropositives and 7% of 120 gB vaccinees lacked A-gB antibodies but had neutralizing activity. Among subjects with delta-gB antibody, there were weak correlations between antibody level and neutralizing titer. These data suggest that antibodies to linear neutralizing gB domains are highly prevalent in naturally-infected individuals and regularly develop in gB vaccinees. However, for some individuals, discontinuous and/or linear epitopes not represented on delta-gB may be more important in the generation of neutralizing responses.

Effect of rapid viral diagnosis on the management of children hospitalized with lower respiratory tract infection.

BACKGROUND: Although rapid viral tests are commonly used in children with lower respiratory tract infection, their effect on patient management has not been studied. OBJECTIVES: To examine how physicians utilize an enzyme immunoassay for respiratory syncytial virus (RSV EIA) and a centrifugation-enhanced cellular immunofluorescence assay for multiple viral pathogens [viral respiratory panel (VRP)] in children hospitalized with respiratory illness; to determine the effect of testing on length of stay, antibiotic use and costs; and to determine physician attitudes toward RSV testing. DESIGN AND SETTING: Prospective study and survey at a large children's hospital. PATIENTS: Previously healthy children < 24 months of age consecutively admitted between January 1 and February 11, 1995, with symptoms of lower respiratory tract infection. RESULTS: Of 200 patients 160 were tested by RSV EIA; 92 were positive and 68 were negative. Tested children were younger, more tachypneic and more likely to require oxygen than those not tested. Overall the length of stay was similar in RSV-positive and -negative patients. Although equal proportions of each group were given antibiotic therapy, RSV-positive children received antibiotic therapy for fewer days than RSV-negative children (median 2 vs. 3 days; P = 0.0387). However, a crude cost analysis did not support a strategy of testing all bronchiolitis patients for RSV. Sixty-five of the 68 RSV-negative children were tested for RSV and other pathogens by VRP. In 55 cases the results were not available until after patient discharge and could not have influenced their management. One hundred three physicians caring for children in the study were surveyed. Of 75 respondents almost all thought that RSV EIA results influenced their management of patients and were important to parents. CONCLUSIONS: Most children hospitalized with symptoms of lower respiratory tract infection were tested for viral pathogens. The VRP provided little clinically useful information. In contrast RSV EIA results may have been used by clinicians to make antibiotic decisions. Physicians felt that rapid testing for RSV was important.

Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells.

Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp13O into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp 130 remained completely sensitive to endo-beta-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp 130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.

Ontogeny of glycoprotein gB-specific antibody and neutralizing activity during natural cytomegalovirus infection.

The envelope glycoprotein gB (gpUL55) is a candidate for inclusion in subunit cytomegalovirus (CMV) vaccines, although data on gB antibody responses after natural infection are limited. [35S]-labeled gB was partially purified from cells infected with an adenovirus recombinant expressing gB and used in radioimmunoprecipitation assays to characterize responses in solid organ transplant recipients with primary (n = 11) or secondary (n = 8) CMV infection. Seropositive transplant patients without evidence of infection (n = 5) and healthy seroconverters (n = 7) were also studied. gB antibody developed concurrently with CMV-specific IgG, IgM, and neutralizing activity in transplant patients with primary infection. Sustained boosts in gB antibody were seen in patients with secondary infection, and healthy seroconverters developed early gB responses. These data imply that gB antibody is an integral part of the humoral response to CMV infection, and, in view of experimental data regarding immunogenicity, support a role for gB in subunit vaccines.

Rapid diagnosis of respiratory viral infections by using a shell vial assay and monoclonal antibody pool.

We compared the detection of seven respiratory viruses by using a commercially available monoclonal antibody pool in a 2-day shell vial assay with that by using standard cell culture with respiratory syncytial virus (RSV) enzyme-linked immunosorbent assay (ELISA)-negative nasal secretions from hospitalized children. We found 179 respiratory virus isolates by either method in 675 specimens. Overall, the shell vial assay detected 147 of 179 (79%) of the positives after 2 days; cell culture detected 148 of 179 (80%) after a mean incubation period of 7.6 days (range, 1 to 14 days). The sensitivity of the shell vial assay was 78% for RSV, 94% for influenza B virus, 83% for adenovirus, and 80% for parainfluenza viruses. The sensitivity of the cell culture was 70% for RSV, 79% for influenza B virus, 90% for adenovirus, and 89% for parainfluenza viruses. The 2-day shell vial assay allowed the detection of respiratory viruses in a clinically relevant time frame and rapidly detected RSV in specimens lacking RSV antigen by ELISA.

Antibodies to recombinant-derived glycoprotein B after natural human cytomegalovirus infection correlate with neutralizing activity.

Glycoprotein B (gB) of human cytomegalovirus (HCMV) was partially purified by lentil-lectin column chromatography from cells infected with an adenovirus-gB recombinant. This antigen, which contained specifically reactive proteins of approximately 130 and 55 kDa, was used to investigate gB antibody levels after natural HCMV infection in 48 individuals. All sera had IgG antibody to gB as detected by radioimmunoprecipitation (RIP) assays. Quantitative RIP showed a strong correlation between gB antibody and neutralizing activity (r = .74, P less than .001) but a weak correlation between gB antibody and total HCMV-specific IgG (r = .36, P less than .02). When gB antibody was specifically absorbed from 20 serum specimens, neutralizing antibody titer was reduced a median of 48% (range, 0-98%). These data confirmed that antibodies to gB are a large component of the neutralizing antibody response to HCMV and support a role for this protein in the development of subunit vaccines.

Oral habilitation of the child with no response on brainstem audiometry.

Children without an auditory brainstem evoked response can benefit from an auditory-oral hearing habilitation program. This study focuses on 31 such children enrolled at the Houston School for Deaf Children. Eleven had excellent auditory-oral skills, with language ability equal to normal-hearing children of a similar age, and speech easily intelligible to a familiar listener. A statistically significant factor associated with success was hearing acuity at 250 Hz. Other important factors included age at enrollment at the school, duration of enrollment, degree of parental support, and absence of middle ear disease.

Educational aspects of cochlear implants in children.

An overview of deaf education as it relates to the needs of the cochlear implant child is given. More specific information concerning the developmental approach to successful listening is described. The progress of specific youngsters is discussed following application of this "approach".