Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies

doi:10.1016/0166-0934(94)90015-9

Journal of Virological Methods

Volume 46, Issue 1, January 1994, Pages 39-50

https://doi.org/10.1016/0166-0934(94)90015-9 Get rights and content

Abstract

We isolated and characterized from a λgt11 expression library clones expressing portions of human cytomegalovirus (HCMV)-p52. This non-structural viral protein is encoded by UL44 and is known to be one of the best IgM reactive antigens. The reactivity of these clones was studied with human antibody and the gene fragment coding for the most immune-reactive portion of p52 (aa 202–434) was cloned in a prokaryotic expression vector, pROS, which overexpresses the antigen as a fusion protein to a truncated molecule of β-galactosidase.

References (23)

W.N. Burnette
‘Western Blotting’: Electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A
Anal. Biochem.
(1981)
W. Gibson
Protein counterparts of human and simian cytomegaloviruses
Virology
(1983)
L. Pereira et al.
Polymorphism of human cytomegalovirus Glycoproteins characterised by monoclonal antibodies
Virology
(1984)
D.J. Braun et al.
Application of denatured, electrophoretically separated and immobilized lysates of Herpes simplex virus infected cells for detection of monoclonal antibodies and for the study of the properties of viral proteins
J. Virol.
(1983)
M.S. Chee et al.
Analysis of the protein content of the sequence of human cytomegalovirus strain AD169, p.125–169
S. Ellinger et al.
Cleavage of prokaryotically expressed human immunodeficiency virus fusion proteins by Factor Xa and application in Western Blott (Immunoblott) assays
J. Clin. Microbiol.
(1989)
P.D. Griffiths
The presumptive diagnosis of primary cytomegalovirus infection in early pregnancy by means of a radioimmunoassay for specific IgM-antibodies
Br. J. Obst. Gynecol.
(1981)
M. Ho
Cytomegalovirus. Biology and Infection, Current Topics in Infectious Disease
(1991)
T.V. Huynh et al.
Constructing and screening cDNA libraries in λgt10 and λgt11
V. Knez et al.
Cytomegalovirus specific IgM and IgG response in humans studied by radioimmunoassay
J. Immunol.
(1976)

M.P. Landini

Humoral immune responses to Cytomegalovirus proteins

Rev. Med. Virol.

(1992)

Cited by (21)

Human cytomegalovirus infection: Diagnostic potential of recombinant antigens for cytomegalovirus antibody detection
2001, Journal of Virological Methods
Recombinant antigen-based enzyme immunoassays (EIAs) for the detection of human cytomegalovirus (HCMV) specific antibody are believed to yield a higher sensitivity and specificity than virus lysate EIAs. The aim of the present study was to evaluate the accuracy of newly established HCMV assays (Copalis CMV Multiplex, Sorin; Cobas Core CMV IgG and IgM EIAs, Roche Diagnostics; Anti-HCMV recombinant IgG, gB-IgG, IgM and IgA, Biotest; and ETI-CYTOK-G PLUS and M reverse PLUS, Sorin) based on recombinant antigens and/or virus lysate for laboratory diagnosis of HCMV infection. For the assessment of sensitivity, follow-up samples from patients suffering from active HCMV infection were tested. Testing a large number of potentially interfering samples challenged the specificity of the assays. There was no statistically significant difference in the performance of HCMV IgG assays. The results were more heterogeneous for the detection of serological markers of active infection (HCMV IgM, HCMV IgA and anti-CM2). The sensitivities of the different assays ranged between 40.5 and 71.4%. A variable number (17.8–1.7%) of false-positive results were obtained among potentially interfering serum samples. Two of the recombinant antigen based assays showed a high degree of interference with EBV VCA-IgM-positive sera. The best performance was achieved with ETI-CYTOK-M reverse PLUS since it combined the highest sensitivity with specificity. Commercially available assays based on recombinant antigens showed, overall, a poorer performance than the virus lysate EIA.
Humoral Immune Response to Proteins of Human Cytomegalovirus Latency-Associated Transcripts
2000, Biology of Blood and Marrow Transplantation
Latent human cytomegalovirus (CMV) infection of hematopoietic progenitor cells is associated with the presence of latency-associated transcripts that may express 6 proteins larger than 44 amino acids in size (open reading frame [ORF] 55, ORF45, ORF94, ORF59, ORF154, ORF152/UL124). The serologic response to these proteins was evaluated in healthy seropositive individuals as well as in individuals undergoing active CMV infection. Individual recombinant GST-fusion proteins, prepared from bacteria, were found by enzyme-linked immunosorbent assay to be recognized by between 8% and 44% long-term healthy seropositive individuals, with ORF94 and ORF55 being the most broadly and significantly recognized. Although nearly all of serum samples (85%) recognized at least 1 of these proteins, none reacted with all 6. Patterns of antibody prevalence to these proteins in long-term seropositive individuals were similar to many antigens expressed during productive replication (IE1, ppUL57, ppUL83/pp65), but none were broadly detected by a majority of individuals, a characteristic of only a few productive-phase antigens, including ppUL44/ICP36 and ppUL32/pp150. Consistent with prevalence in long-term seropositive individuals, commercial preparations of pooled human gamma globulin were also found to recognize latency-associated proteins. Serologic reactivity to latency-associated proteins was slow to develop following primary infection, in a pattern distinct from any of the characterized replication-phase proteins tested here, and was boosted late after secondary infection or reactivation in solid-organ transplant recipients without showing a correlation with viremia or disease. These results provide evidence that proteins expressed from the latent region during natural infection exhibit immunogenicity comparable with most other characterized viral antigens, although the narrow response to individual latency-associated proteins likely precludes their use in serologic assays to investigate clinical correlates or outcome in transplant recipients.
Biol Blood Marrow Transplant 2000;6(2):100-8.
Intracellular production of a major cytomegalovirus antigenic protein in the methylotrophic yeast Pichia pastoris
1996, Gene
We have previously shown that single or multiple epitopes of the major human cytomegalovirus (HCMV) antigens, produced as fusion proteins in prokaryotes can be valuable diagnostic material in the serology of HCMV infection. In this work we moved to a eukaryotic system, to produce one of the most immunogenic HCMV antigens, ppUL44 (also called pp52 due to its apparent molecular size on acrylamide gels), as a non-fusion protein, in an attempt to eliminate some non-specific reactivity of human sera with bacterial carrier proteins. We expressed the DNA encoding ppUL44 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. Good levels of intracellular, soluble pp52 were produced. We observed an indistinguishable pattern of the yeast pp52 from the viral native protein in immunoblotting and a good reactivity with human sera.
Specific IgE detected by ELISA and immunoblot after human cytomegalovirus infection (HCMV) in renal transplant (RT) recipients
1996, Clinical and Diagnostic Virology
Background: Specific HCMV IgE response has been reported by some authors, and was proposed as a valuable virologic marker of CMV infection.
Objectives: we evaluated specific HCMV IgE in renal transplant patients with active (primary and secondary) HCMV infection with special interest to symptomatic infections.
Study design: Specific IgE was tested retrospectively by ELISA and immunoblot (IB) on sera of 55 RT patients who were followed before and after transplantation with virologic markers of CMV infection.
Results: Total serum IgE levels were similar in control group and in patients with primary and secondary HCMV infections. Anti-CMV specific IgE response by ELISA was more frequently found in patients with primary infection (76.9%) than in patients with secondary infection (47.1%). These specific IgE reacted on immunoblot with a 150 kDa protein in 84.6% of patients with primary infection and 94.1% with secondary infections; and reacted with rp52 (pUL44) in 76.9% of primary infection and 47.1% of secondary infection.
Conclusions: Anti-CMV specific IgE tested by immunoblot and ELISA is a marker of CMV infection. It was clearly detected in cases of active infection (primary and secondary) and was present in cases with severe CMV clinical manifestations. In contrast, anti-CMV specific IgE, was consistently negative among healthy blood donors. This is the first report of CMV proteins detected by IgE immunoblot.
Characterisation of a human antibody that potentially links cytomegalovirus infection with systemic lupus erythematosus
2019, Scientific Reports
The use of pp150 and gp116 synthetic peptides in the detection of CMV antibodies
2010, Iranian Red Crescent Medical Journal

View all citing articles on Scopus

View full text

Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies

Abstract

Anal. Biochem.

Virology

Virology

Application of denatured, electrophoretically separated and immobilized lysates of Herpes simplex virus infected cells for detection of monoclonal antibodies and for the study of the properties of viral proteins

J. Virol.

Analysis of the protein content of the sequence of human cytomegalovirus strain AD169, p.125–169

Cleavage of prokaryotically expressed human immunodeficiency virus fusion proteins by Factor Xa and application in Western Blott (Immunoblott) assays

J. Clin. Microbiol.

The presumptive diagnosis of primary cytomegalovirus infection in early pregnancy by means of a radioimmunoassay for specific IgM-antibodies

Br. J. Obst. Gynecol.

Cytomegalovirus. Biology and Infection, Current Topics in Infectious Disease

Constructing and screening cDNA libraries in λgt10 and λgt11

Cytomegalovirus specific IgM and IgG response in humans studied by radioimmunoassay

J. Immunol.

Humoral immune responses to Cytomegalovirus proteins

Rev. Med. Virol.