HIV-1 infection includes an initial acute clinical syndrome typically followed by a prolonged clinical latency and the eventual development of AIDS. A substantial percentage of HIV-1-infected subjects remain disease-free for many years with a low viral load and an intact lymph-node architecture. Moreover, cohorts of HIV-1-exposed individuals who remain free of infection over a long period of viral exposure have been described. The molecular basis of this heterogeneous clinical presentation is unknown; one possibility is that differences in humoral immune responses to yet unrecognized epitopes may contribute to the development of a long-term non progressive state of HIV-1 infection. The aim of this project is to systematically compare the antibody specificities of typically progressive and non-progressive HIV-1-infected subjects. Specifically, we plan to identify the immunogenic epitopes recognized by the antibodies present in these different groups of individuals. To this end, Random Peptide Libraries (RPL) displayed on phages have been utilized. These libraries carry random sequences of nine residues expressed in frame with the pVIII coat protein of f11.1 filamentous phages. Up to 2,700 copies of a single epitope can be displayed on a single phage particle, while the complexity of the libraries is 2 x 10/7 independent clones. Two different libraries have been utilized to identify epitopes specifically recognized by serum immunoglobulins of subjects affected by different clinical degrees of HIV-1 infection. An original methodology has been developed whereby HIV-1-specific epitopes were isolated by using sequential steps of purification: a) immunoaffinity selection of phages by HIV-positive sera immunoglobulins bound to magnetic beads; b) immunoscreening of the eluted phages transferred on nitrocellulose filters; c) selection of the phage clones by ELISA; d) purification of the phage colonies and sequencing of the inserted peptide. The results are as follows: from the initial input of 2 X 10/13 phages, 625 phage colonies were selected for their reactivity with sera from 30 HIV-positive subjects and counter-screened against 50 sera from HIV-negative healthy subjects. 10 clones were selected for their consistent association with HIV-positive sera and the absence of reactivity with control sera. These clones were amplified and sequenced. The sequence analysis revealed that two clones carry a peptide belonging to a region of HIV-1 Env and Gag protein, respectively, while the remaining peptides likely represent discontinuous epitopes. The purified phages have been injected into mice to test for their immunogenicity. Moreover, the selected phages have been utilized to immunoaffinity purify the epitope-specific antibodies from HIV-infected subject. These HIV-specific antibodies are currently being tested for the capacity to interfere in vitro with HIV-1 infection of autologous or heterologous T lymphocytes.