Cloning and characterization of major antigenic determinants of human cytomegalovirus Ad169 seen by the human immune system

Abstract

Starting from a cosmid library of HCMV Ad169-DNA random fragments of DNA were generated. Fragments about 200 to 600 bp in length were selected and cloned into open reading frame (ORF) expression vectors to create ORF-libraries that represent either the entire viral genome or defined subregions. About 120,000 clones were isolated and screened immunologically for the synthesis of fusion proteins consisting of an antigenic peptide encoded by the CMV sequence coupled to a truncated E. coli beta-galactosidase molecule. Anti-CMV sera raised in animals as well as human hyperimmune globulin were used for colony screening. Distinct sets of antigenic fusion proteins were recognized by different antisera. Ten of the clones giving strong reactions with human immune sera were mapped on the CMV genome and the sequences of the CMV inserts determined. Antibodies against fusion proteins were raised in mice or rabbits to identify the corresponding CMV proteins. Antigenic fusion proteins described here were recognized by most individual human CMV immune sera tested. They allow determination of the humoral immune response to defined determinants and may therefore be particularly useful in diagnosis and vaccine development.

Stably expressed antisense RNA to cytomegalovirus UL83 inhibits viral replication

“…A construct containing the region coding pp71 from amino acids 434 to 495 (pStuB32) was the generous gift of W. Lindenmaier (20).…”

“…Hybridization with the pp65-specific probes was carried out as described above. As probes, we used (i) UL83 ORF DNA described above; (ii) [ 32 P]UTP-labeled antisense probe (which recognizes the sense RNA) generated by using the 3Ј SP6 promoter; and (iii) [ 32 P]UTP-labeled sense probe (which recognizes the antisense RNA) generated by using the 5Ј T7 promoter of T7SP6-pp65 by in vitro transcription using a RiboMax kit (Promega) according the manufacturer's specifications after linearization of the plasmid with BamHI and XhoI, respectively; and (iv) a pp71-specific probe (20); and (v) a glyceraldehyde 3-phosphate dehydrogenase-specific probe, both 32 P labeled by random priming.…”

Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes

“…Three overlapping ␤-galactosidase (␤gal) pp65 fusion proteins were chosen (Fig. 1), which are recognized by the humoral immune response [Landini et al, 1990;Lindenmaier et al, 1990]. The three pp65 fusion proteins (i) C74 (amino acids [aa] 297-458), (ii) C35 (aa 303-476), and (iii) C47 (aa 389-561) cover the C-terminal 265 amino acids of the pp65 sequence [Chee et al, 1990].…”

Anti-neutrophil cytoplasmic antibodies (ANCA) from patients with systemic vasculitis recognize restricted epitopes of proteinase 3 involving the catalytic site

“…Linear peptides can identify regions of antigens important in binding of antibodies [16, 29]. The SPOT system is particularly suited to this approach, as it allows multiple peptides to be synthesized onto a cellulose membrane, and these can be repeatedly probed with different antibodies [30–32]. This system has been successfully used in our laboratory to define epitopes of the Goodpasture antigen [33].…”