Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus.

A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR/TGGE) has been established for the quantification of human cytomegalovirus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wildtype (wt) amplimers by temperature gradient gel electrophoresis (TGGE). The number of HCMV target sequences could be precisely determined within wt/st ratios of 0.1 to 10. With 50 copies of the st sequence the detection limit of nPCR/TGGE was found to be five to l0 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification offl-globin target sequences and calculation of the ratio of HCMV copies/fl-globin copies. Serial peripheral blood leukocyte specimens of 17 renal allograft recipients positive in a qualitative nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood donors served as negative controls. Positive results were obtained by nPCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia and positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/fl-globin ratios (10000 to 8000 copies HCMV/ 106 copies fl-globin) were found in transplant recipients experiencing acute clinically symptomatic HCMV infection. HCMV DNA levels in asymptomatic patients ranged from 900 to 200 copies HCMV/10 ~ fl-globin.

Section: Methodsmentioning

confidence: 99%

“…* Localization of HCMV and fl-globin primers indicated according to Cranage et al (1986) and Orkia & Kazazian (1984).…”

confidence: 99%

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

Schäfer

Braun

Möhring

et al. 1993

“…A major target of the immune response to CMV infection is glycoprotein B (gB), a constituent of the virion envelope and a homologue of herpes simplex virus 1 gB (Chee et al, 1990). The CMV gB gene has been mapped in the genomes of strain AD169 (Cranage et al, 1986;Mach et al, 1986) and Towne Spaete et al, 1988). This glycoprotein is produced as a full-length precursor, which is glycosylated (Britt & Auger, 1986;Pereira et al, 1984;Rasmussen et aL, 1985bRasmussen et aL, , 1988 and proteolytically cleaved between residues 460 and 461 after its synthesis (Spaete et al, 1988(Spaete et al, , 1990.…”

mentioning

confidence: 99%

The amino terminus of human cytomegalovirus glycoprotein B contains epitopes that vary among strains

Basgoz

Qadri

Navarro

et al. 1992

We mapped three antigenic domains of continuous epitopes on human cytomegalovirus (CMV) glycoprotein B (gB) by reacting a panel of independently derived monoclonal antibodies with deletion mutants expressed transiently in COS-1 cells. One of these antigenic domains, DC2, maps in the last 75 amino acids of the carboxy terminus. These epitopes are conserved in strains Towne and AD169, as well as in 19 clinical CMV isolates. ELISAs of DC2-reactive antibodies with a set of overlapping synthetic oligopeptides from the carboxy terminus showed that the epitopes of antibodies CH405-1 and CH421-5 map between amino acids 833 and 852 and that the epitope of antibody CH28-2 maps between amino acids 878 and 898. These linear epitopes were grouped into domain DC3. The third antigenic domain, DClv, maps at the aminoterminal end of CMV strain AD169 gB but is not contained in strain Towne or in 17 of 19 clinical isolates. Epitopes in this domain are likely to map between amino acids 28 and 67, an area where differences occur in the nucleotide sequence of the gB genes from AD169 and Towne. Analysis of CMVinfected cells by flow cytometry with antibodies to the amino-and carboxy-terminal domains revealed that the amino terminus of gB is extracellular and that the carboxy terminus is not exposed on the cell surface.

“…The HCMV gB gene encodes a 130K precursor protein (gpl30) which is thought to be cleaved by a cellular enzyme, giving rise to a C-terminal 55K glycoprotein, designated gp55 (Spaete et al, 1988). The Mr of the gB complex has been shown to vary (Pereira et al, 1984;Britt, 1984;Britt & Auger, 1986;G6ncz61 et at., 1986;Landini et al, 1987;Mach et al, 1986;Cranage et al, 1986), but its abundance in the virion and immunological importance make it likely that the same glycoprotein was detected in all investigations.…”

Section: Introductionmentioning

confidence: 95%

“…Human and murine monoclonal antibodies (MAbs) showing HCMV neutralizing activity were shown to recognize both gpl30 and gp55 (Masuho et at., 1987 ;Cranage et al, 1986;Nowak et al, 1984;Utz et al, 1989). In a study of human volunteers immunized with the attenuated HCMV Towne strain and challenged with the virulent Toledo strain, all sera showing neutralizing activity also reacted with the 55K to 58K protein derived from gB (G6ncz61 et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Characterization of a Major Antigenic Region on gp55 of Human Cytomegalovirus

Silvestri

Sundqvist²,

Rudén³

et al. 1991

A major antigenic region localized to the C-terminal part of the 55K glycoprotein of human cytomegalovirus (HCMV) was mapped using synthetic peptides. Analysis of the region with six sera from healthy anti-HCMV seropositive blood donors showed that the length of the reactive sequence varied between four and eight amino acids (aa). The shortest sequence recognized was VTSG (aa 793 to 801 of the 130K precursor protein), but most sera required the three or four residues C-terminal to this to react, giving a major site ofVTSGSTKD (aa 798 to 805). Using peptides immobilized on polyethylene pins, the importance of both interior (between aa essential for the binding of an antibody) and extension spacer residues was evident. Free peptides containing the reactive sequence were prepared for use in a conventional ELISA and optimal pH conditions for coating were determined. The best results were achieved at pH 2 to 3, which is in agreement with the advantageous net charge of these peptides at this low pH. Anti-HCMV positive sera showed a sensitivity of approximately 50% for both peptides on polyethylene pins and peptides coated onto plates.