Epitope analysis of human cytomegalovirus glycoprotein complexes using murine monoclonal antibodies

Lussenhop, Nancy; Goertz, Ron; Wabuke-Bunoti, Mel; Gehrz, Richard C.; Kari, B.

doi:10.1016/0042-6822(88)90549-1

Cited by 43 publications

(45 citation statements)

References 16 publications

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“…This assay was done as previously described (Lussenhop et al, 1988). Briefly, purified Towne strain HCMV or whole gC-I were coated onto Immunolon 96-well microtitre plates.…”

Section: Methodsmentioning

confidence: 99%

“…Virus was labelled with either [3H]arginine (Arg) or [3H]glucosamine (GIcN) (Amersham). MAbs to gp52 (Lussenhop et al, 1988) and gp93-130 (Karl et al, 1990) were generated and characterized as described.…”

Section: Methodsmentioning

confidence: 99%

“…Previously we mapped the epitopes on gp52 recognized by several of our MAbs (Lussenhop et al, 1988). Using a competitive binding ELISA, we were able to assign the epitopes recognized by the MAbs into three domains based on the ability of the MAbs to inhibit the binding of other MAbs.…”

Section: Introductionmentioning

confidence: 99%

“…Domain I contained epitopes recognized by MAbs 41C2, 26B11 and 39Ell. These epitopes were both continuous (41C2 and 39Ell) and discontinuous (26B 11 (Lussenhop et al, 1988). Domain III contained a discontinuous epitope recognized by MAb l lB4.…”

Section: Introductionmentioning

confidence: 99%

“…Because gC-I complexes are large disulphide-linked structures they have the potential to contain many continuous and discontinuous epitopes. Both gp52 (Lussenhop et al, 1988) and gp93-130 (Karl et al, 1990) contain epitopes recognized by neutralizing murine monoclonal antibodies (MAbs). However, most MAbs made using whole virus as antigen have recognized gp52 (Banks et al, 1989;Karl et al, 1986;Britt, 1984;Rasmussen et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

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Biochemical and Immunological Analysis of Discontinuous Epitopes in the Family of Human Cytomegalovirus Glycoprotein Complexes Designated gC-I

Kari

Gehrz

1991

Journal of General Virology

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The envelope of human cytomegalovirus contains a family of disulphide-linked glycoprotein complexes designated gC-I which contain two glycoproteins of 52 000 Mr (gp52) and 93000 to 130000 Mr (gp93-130). Epitopes recognized by several of our gC-I gp52-specific monoclonal antibodies (MAbs) were previously assigned to three domains based on reactivity with gC-I in a competitive binding assay. In this report, we have used additional gC-I MAbs to characterize three distinct discontinuous epitopes in the gC-I complexes. Two of these epitopes were in Domain I and one in Domain III. These epitopes were resistant to proteolysis, heat denaturation and SDS treatment. However, the discontinuous epitopes were lost after reduction of disulphide bonds. After digestion of gC-I complexes with chymotrypsin, two fragments of 43 000 (43K) Mr and 34000 (34K) Mr were obtained which contained all discontinuous and continuous epitopes recognized by our gp52 MAbs. The Mr of these fragments could not be reduced further by longer digestion or by use of other proteases such as trypsin or pronase. The 43K fragment contained N-linked oligosaccharides not detected in the 34K fragment. These oligosaccharides may have prevented a complete proteolytic digestion so that the 34K fragment was not always obtained. It was established that 80 to 90% of the mass of these fragments was contributed by gp52. Thus the discontinuous epitopes were composed primarily of gp52 and not gp93-130.

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“…This assay was done as previously described (Lussenhop et al, 1988). Briefly, purified Towne strain HCMV or whole gC-I were coated onto Immunolon 96-well microtitre plates.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Biochemical and Immunological Analysis of Discontinuous Epitopes in the Family of Human Cytomegalovirus Glycoprotein Complexes Designated gC-I

Kari

Gehrz

1991

Journal of General Virology

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Humoral immune response to functional regions of human cytomegalovirus glycoprotein B

et al. 1997

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Sera from patients with primary human cytomegalovirus (HCMV) infections, both acute and convalescent phase, and from HCMV-seropositive healthy subjects were analyzed to determine whether the sera would recognize antigenic domains on HCMV glycoprotein B (gB) that function in virion infectivity and spread of virus from cell to cell. The intact gB molecule, amino-terminal derivatives of different lengths, and internal deletion derivatives were expressed in eukaryotic cells and reacted by immunofluorescence with the sera. All convalescent-phase sera and most sera from healthy seropositive individuals reacted with full-length gB and with an amino-terminal derivative containing 687 amino acids (aa), gB-(1-687); approximately half of the sera recognized an amino-terminal derivative of 447 aa, gB-(1-447), and one-third reacted with the shortest deletion derivative of 258 aa, gB-(1-258). Of the acute-phase sera, 77% recognized intact gB and gB-(1-687), 32% recognized gB-(1-447), and 14% recognized gB-(1-258). Deletion of aa 548 to 618 dramatically reduced the percentage of reactive sera, whereas deletion of aa 411 to 447 had a minor impact on reactivity of sera. To investigate the epitope specificity of human antibodies to gB, we carried out competition experiments between human sera with neutralizing activity and selected monoclonal antibodies (mAbs) to conformational epitopes on gB. We found that antibodies in human sera preclude syncytium formation in UB15-11 glioblastoma cells constitutively expressing gB and compete with certain murine mAbs that block virus entry into cells and transmission of infection from cell to cell. Our results show that HCMV-immune human sera contain antibodies to functional regions on gB, and the abundance of these antibodies in convalescent-phase sera suggests that they may play a central role in limiting dissemination of virus in the host.

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An ELISA using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus

Kropff

Landini

Mach

1993

Journal of Medical Virology

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Two prokaryotically expressed fusion proteins encompassing amino acids 484-650 (AD-1) and 27-100 (AD-2) of glycoprotein gp58/116 of human cytomegalovirus (HCMV) were purified from E. coli lysates and used in ELISA to determine antibody levels in human sera. The specificity of the test was established by comparison of 116 randomly selected sera with commercially available HCMV-ELISA tests. The recombinant polypeptides were then used for the analysis of antibody titers in 112 human sera and were compared to the capacity to neutralize HCMV. A strong correlation between the neutralization titer and antibody levels against AD-1 and a weaker correlation for AD-2 was observed. Of 29 sera with a high neutralization titer (> 1:128), 96% and 62% were positive for AD-1 and AD-2, respectively, while 44% and 19% were positive in sera with low neutralization titer (< 1:8). Serum pools prepared from human sera selected on the basis of recognition of the recombinant antigens had a 10-fold higher neutralization capacity than pools prepared from sera with a high titer in commercially available HCMV tests. A synchronous increase in neutralization capacity and titer against recombinant antigens was observed in transplant patients.

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Epitope analysis of human cytomegalovirus glycoprotein complexes using murine monoclonal antibodies

Cited by 43 publications

References 16 publications

Biochemical and Immunological Analysis of Discontinuous Epitopes in the Family of Human Cytomegalovirus Glycoprotein Complexes Designated gC-I

Biochemical and Immunological Analysis of Discontinuous Epitopes in the Family of Human Cytomegalovirus Glycoprotein Complexes Designated gC-I

Humoral immune response to functional regions of human cytomegalovirus glycoprotein B

An ELISA using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus

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