title: Construction of polyepitope fusion antigens of human cytomegalovirus ppUL32: reactivity with human antibodies.
abstract: We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides, can be valuable diagnostic material in the serology of HCMV infection (M. P. Landini, M. X. Guan, G. Jahn, W. Lindenmaier, M. Mach, A. Ripalti, A. Necker, T. Lazzarotto, and B. Plachter, J. Clin. Microbiol. 28:1375-1379, 1990; M. P. Landini, T. Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa, J. Clin. Microbiol. 27:2324-2327, 1989; A. Ripalti, M. P. Landini, E. S. Mocarski, and M. La Placa, J. Gen. Virol. 70:1247-1251, 1989). In this work we addressed the question of whether the expression of more than one linear epitope on a single fusion protein could increase the reactivity of genetically engineered antigenic material with human antibody. To answer this question we fused sequences expressing two different epitopes contained in the basic phosphoprotein of 150 kDa encoded by UL32 (M. S. Chee, A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, T. Cerny, T. Hornsel, C. A. Hutchinson, T. Kouzarides, J. A. Martignetti, and B. G. Barrell, Curr. Top. Microbiol. Immunol. 154:125-169, 1990; G. Jahn, T. Kouzarides, M. Mach, B.-C. Scholl, B. Plachter, B. Traupe, E. Preddie, S. C. Satchwell, B. Fleckenstein, and B. G. Barrell, J. Virol. 61:1358-1367, 1987), ppUL32, which was repeatedly shown to be the strongest immunogen present in the viral particle. We also made fusions with sequences expressing a single epitope repeated once, twice, or three times. The different fusion proteins were tested with HCMV-positive human sera. We found that fusion proteins expressing different epitopes together were recognized by a larger number of serum specimens and with more intense reactions in Western blot (immunoblot) experiments. We also found evidence that expression on the same polypeptide of the two distinct epitopes produced a stronger antigen than the mere addition of two fusion proteins which each carried one copy of one of these epitopes. Furthermore, we found that while the same epitope expressed two or three times on the same fusion protein was not better recognized by immunoglobulin G than the single epitope, immunoglobulin M reactivities to the double and triple epitopes were enhanced.
authors.affiliation: Department of Microbiology, School of Medicine, University of Bologna, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Ripalti
authors.forename: Q
authors.initials: Q
authors.lastname: Ruan
authors.forename: M C
authors.initials: MC
authors.lastname: Boccuni
authors.forename: F
authors.initials: F
authors.lastname: Campanini
authors.forename: G
authors.initials: G
authors.lastname: Bergamini
authors.forename: M P
authors.initials: MP
authors.lastname: Landini
mesh_terms: D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000276:immunology; D004279:DNA, Viral / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D004279:DNA, Viral; D000939:Epitopes; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins