Cindy L. Jellis

Defining critical residues m the epitope for a hiv-neutralizing monoclonal antibody using phage display and peptide array technologies

A peptide display library [Scott and Smith, Science 249 (1990) 386-390] was constructed that expressed 1.5 x 10(8) unique 20-amino-acid (aa) peptides fused to the N-terminus of the pIII coat protein of filamentous phage fd. This phage display library (PDL-20) was prepared using a degenerate oligodeoxyribonucleotide designed to minimize bias towards most aa. Characterization of the PDL-20 showed that all aa were present at the expected frequency and that there was no positional bias. Screening of this library with a HIV-1 isotype MN envelope reactive monoclonal antibody (mAb 58.2) using two different panning procedures showed that the biopanning technique was sensitive to one phage in 10(8). Analysis of peptide sequences from panning the mAb identified a core antibody recognition sequence of four aa residues (GPGR) and two preferred flanking residues on either side. This epitope occurred at various locations within the random aa segment demonstrating an absence of positional or nearest neighbor effects. Parallel panning experiments using an array of 266 synthetic peptides identified an epitope similar to that defined by the phage display library.

Genomic organization of the gene coding for the costimulatory human B-lymphocyte antigen B7-2 (CD86)

The generation of an antigen-specific T-cell response requires that the T lymphocyte receive two signals from the antigen presenting cell. The specificity of this response is provided by antigen presented to the T lymphocyte and involves stimulation of the T lymphocyte via the T-cell receptor (TCR)/CD3 complex. The second, or costimulatory signal, can be provided by ligation of the B-lymphocyte activation antigens B7-1 (CD80) and B7.2 (CD86) to TCR antigen CD28. The cDNAs for both CD80 and CD86 have been isolated and are predicted to encode type 1 membrane proteins of the immunoglobulin (Ig) superfamily. The predicted protein is composed of a signal peptide followed by two Ig-like extracellular domains, a transmembrane domain, and a cytoplasmic tail. Here we report that the genomic organization of CD86 reflects its functional structure, and is similar to that oound for CD80. The gene is composed of eight exons which span more than 22 kilobases. The predicted protein functional domains of signal peptide, extracellular IgV- and Ig-like regions, and transmembrane domain coincide with the genomic structure. Two independent sequences had been reported for CD86 cDNA which differed in their 5′-untranslated (UT) regions. We find CD86 exons 1 and 2 correspond to these alternate 5′UT sequences. Splicing of exon 1 or 2 with the signal peptide encoding exon 3 would produce mRNA transcripts complementary to the reported cDNA clones. Exons 4 and 5 correspond to IgV- and IgC-like extracellular domains, respectively. Exon 6 encodes the transmembrane region and beginning of the cytoplasmic tail. Exons 7 and 8 encode the remainder of the cytoplasmic tail and 3′UT sequences.