Expression of the Human Parvovirus B19 Protein Fused to Protein A in Escherichia coli: Recognition By IgM and IgG Antibodies in Human Sera

Morinet, F.; d'Auriol, L; Tratschin, Jon-Duri; Galibert, Francis

doi:10.1099/0022-1317-70-11-3091

Cited by 36 publications

(14 citation statements)

References 31 publications

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“…Cotmore et al (1986) did not investigate their potential as diagnostic antigens, whereas Sisk & Berman (1987) used Western blotting, a technique not suitable for large-scale screening. Morinet et al (1989) obtained soluble expression of a Protein A fusion protein containing the same B 19 sequences as that found in our fl-galactosidase fusion protein encoded by pDEV44. Although an IgM ELISA was developed, the yield of protein in this system was very low and the product consisted mainly of breakdown fragments.…”

Section: Discussionmentioning

confidence: 97%

“…However, except in the report of Morinet et al (1989), these proteins have not been fully exploited for diagnostic use, probably owing to their insoluble nature. Cotmore et al (1986) did not investigate their potential as diagnostic antigens, whereas Sisk & Berman (1987) used Western blotting, a technique not suitable for large-scale screening.…”

Section: Discussionmentioning

confidence: 99%

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The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens

Rayment

Crosdale²,

Morris³

et al. 1990

Journal of General Virology

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We have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B 19 in Escherichia coli. These include native VP1 (8410 and VP2 (58K) proteins and also fusions to flo galactosidase containing differing amounts of the amino terminus of the VP1/2 polypeptide. Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble. This soluble polypeptide, p132, is a/3-galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1. Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples. We conclude that this region contains epitopes which must be prominently exposed on the intact virus. We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples. In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay. The recombinant p132 antigen is efficiently produced and readily purified from E. coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens

Rayment

Crosdale²,

Morris³

et al. 1990

Journal of General Virology

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“…Expression of novel proteins in baculovirus can be easily repeated once the recombinant viruses have been constructed; in addition, the quantity of recombinant protein expressed and the ease of cell culture make the baculovirus system more efficient in labor and materials than mammalian cell culture. Although B19 parvovirus proteins have been expressed in bacteria (22,23) and a cyclic peptide based on the virus's sequence has been synthesized (24), assays based on these products have been imperfect, usually due to inability to detect IgM antibody or a general lack of specificity or sensitivity. We recently using a different baculovirus system by Brown and coworkers (25): their recombinant protein functioned like intact virions in an immunofluorescent assay and in immunoblot analysis of sera (however, they did not demonstrate capsid formation).…”

Section: Methodsmentioning

confidence: 99%

Self-assembled B19 parvovirus capsids, produced in a baculovirus system, are antigenically and immunogenically similar to native virions.

Kajigaya

Fujii

Field

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

228

130

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B19 parvovirus is pathogenic in humans, causing fifth disease, transient aplastic crisis, some cases of hydrops fetalis, and acquired pure red cell aplasia. Efforts to develop serologic assays and vaccine development have been hampered by the virus's extreme tropism for human bone marrow and the absence of a convenient culture system. We constructed recombinants containing either the major (VP2) or minor (VP1) structural proteins of B19 in a baculovirus-based plasmid, from which the polyhedrin gene had been deleted; these recombinant plasmids were used to generate recombinant infectious baculovirus. Subsequent infection of insect cells in vitro resulted in high-level expression ofeither B19 VP1 or VP2. Parvovirus capsids were obtained by self-assembly in cell cultures coinfected with either VP1-and VP2-containing baculoviruses or, surprisingly, VP2-containing baculoviruses alone. Empty B19 capsids composed of VP1 and VP2 could replace serum virus as a source of antigen in a conventional inmmunoassay for detection ofeither IgG or IgM antiparvovirus antibodies in human serum. Immunization of rabbits with capsids composed of VP1 and VP2 resulted in production of antisera that recognized serum parvovirus on immunoblot and neutralized parvovirus infectivity for human erythroid progenitor cells. Baculovirus-derived parvovirus antigen can substitute for scarce viral antigen in immunoassays and should be suitable as a human vaccine.B19 parvovirus is the only member ofthe family Parvoviridae pathogenic in humans (1, 2). Acute infection results in fifth disease, a common childhood exanthem that in adults more usually manifests as an arthralgia/arthritis syndrome. In persons with underlying hemolysis, acute infection produces transient aplastic crisis, precipitous anemia due to hypoproliferative erythropoiesis. B19 infection can be persistent (3). In the setting of immunodeficiency, due to congenital, acquired, or iatrogenic immunodeficiency, persistent parvovirus results in chronic pure red cell aplasia. In utero infection causes hydrops fetalis, with fetal death due to severe anemia and congestive heart failure (4).Clinical studies of B19 parvovirus infection have been hampered by limited supplies of viral antigen. The virus has extraordinary tropism for human erythroid progenitor cells and has only been propagated in explanted human bone marrow (5-8), fetal liver (9), and (to a lesser degree) erythroleukemia cells (10). We describe here expression of the virus's structural proteins in a baculovirus system. Large quantities of empty viral capsids were produced, which can substitute for serum virus in clinical assays and stimulate the production of neutralizing antibodies in animals. Only the major protein was required for capsid assembly; the minor capsid protein serves an important role in virus function independent of virion assembly. MATERIALS AND METHODSCell Culture and Virus Stocks. Autographa californica nuclear polyhedrosis virus (AcMNPV) and recombinant viruses were grown in monolayers of Sf9 cells. Sf9 c...

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“…Both of these proteins have been expressed in numerous prokaryotic and eukaryotic expression systems in order to assess their potential use as diagnostic reagents for parvovirus B19 infection (Morinet et al, 1989;Brown et al, 1990). Indeed, O'Neill et al (1995) concluded that both baculovirus-derived parvoviral antigens (VP1 and VP2) were of equivalent potency with respect to antibody detection and that the superior sensitivity of detection obtained with respect to the native virus may be due to the more controlled purification conditions associated with recombinant antigen purification.…”

Section: Introductionmentioning

confidence: 99%

Detection of parvovirus B19 IgM by antibody capture enzyme immunoassay: receiver operating characteristic analysis

Doyle

Kerr

O’Keeffe

et al. 2000

Journal of Virological Methods

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Parvovirus B19 infection can cause severe effects in high-risk groups including pregnant women and immunocompromised individuals. Although serological detection of B19 infection is commonplace, minimal information is available on the absolute performance characteristics of various tests for the detection of B19 IgM. The performance of the first parvovirus B19 IgM enzyme immunoassay to be cleared by the US Food and Drug Administration (FDA) is described. The immunoassay cut-off has been established using receiver operating characteristic (ROC) analysis giving a sensitivity and specificity of detection of 89.1 and 99.4%, respectively. No cross-reactivity is observed with rubella or other viral disease IgM which cause similar symptomologies to parvovirus B19. Multi-site reproducibility studies have shown high immunoassay reproducibility with detection rates (observed/expected result) of 100% for nonreactive specimens (N=324) and strongly reactive (N=403), respectively. Immunoassay reproducibility ranged from 11.76 to 17.46% coefficient of variation for all reactive specimens tested (N= 12) whereby each specimen was assayed a total of 81 times. Parvovirus B19 IgM seroprevalence of 1% was observed in a US blood donor population (N= 399). In the absence of international performance criteria, this study will be of major benefit to the clinical virologist in assessing immunoassay reliability for the detection of recent infection with parvovirus B19.

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Expression of the Human Parvovirus B19 Protein Fused to Protein A in Escherichia coli: Recognition By IgM and IgG Antibodies in Human Sera

Cited by 36 publications

References 31 publications

The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens

The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens

Self-assembled B19 parvovirus capsids, produced in a baculovirus system, are antigenically and immunogenically similar to native virions.

Detection of parvovirus B19 IgM by antibody capture enzyme immunoassay: receiver operating characteristic analysis

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