Combinatorial Peptide Library as an Immunogen

Abstract

For the past few years, combinatorial chemistry has become a rapidly expanding field in medicmal chemistry (1–3). Obtaining large numbers of structurally diverse compounds represents a major apphcation of combinatorial chemistry, and such divergent libraries are used in the identification of possrble candidate molecules, employing various screemng methodologies. We have designed and synthesized a completely different type of combinatorial libraries, referred to as “mixotope” or “convergent libraries.” These libraries, composed of closely related peptides mixture, collectively present to the immune system a defined antigemc determinant together with its variable motifs. A typical example is the principal antigenic determinant susceptible of eliciting neutralizing antibodies to the human immunodeficiency vu-us (HIV-l), the causative agent of acquired immunodeficiency syndrome (AIDS) This antigemc determinant (epitope), located in the third hypervariable domain (V3) of the HIV surface glycoprotem, gp 120, consists of 32–35 amino acids located between two invariant cystemes lurked by a disulfide bridge (4). In this region, differences exceeding 50% can be found between isolates; thus, recombinant proteins and peptides derived from different isolates have been shown in general to induce antibodres that neutralize only particular subtypes of HIV-l. To recruit simultaneously the broadest part of the repertoire capable of recogmzing not only the known isolates, but also the most probable mutants, we have proposed the use of a mixotope designed from the natural variability of the V3 loop sequence (5). Our initial hypothesis was that molecular structures involved in the immune recognition exhibit a much broader recogmtion pattern than usually considered, being able to tolerate vartations in several positions of the epitope. After alignment of a serves of V3 sequences, we have selected in each position the most represented amino acids, with a threshold of 8% In the degenerated positions, each amino acid was represented in equimolar amount, in order to allow the characterization of the construct by determination of the amino acid composition. This first mixotope was 22–25 residues long and contamed a mixture of 7.5 *x 10⁵ closely related peptides that were obtained in a single synthesis by mcorporating simultaneously 1–6 amino acids in each positions.