Pneumocystis (Pc), a fungal opportunistic pathogen of humans, is the causative agent of Pneumocystis pneumonia (PcP), a leading cause of serious illness in immunocompromised people. As an alternative approach to treatment of PcP in HIV+ patients, which typically involves the long-term use of anti-Pc drugs, offers no protection from subsequent infections with Pc, and may lead to the acquisition of drug resistance by the microorganism, we propose to study antibody reactivity to Pc epitopes in HIV+ patients that did or did not have a previous bout of PcP. The Iong term goal is to identify epitopes for following Pc-specific immune reconstitution in HIV+ patients undergoing HAART. The overall goal of this proposal is to compare serum antibody recognition of the major surface glycoprotein (Msg) of Pc in patients that did or did not have a bout of PcP. Msg is a highly variable protein expressed on the surface of Pc which is recognized by the immune system, and may be involved in antigen switching and immune evasion. We will concentrate on the carboxy terminus of Msg (MsgC) which has previously been shown to differentiate between PcP+ and PcP- patients. The specific aims of this proposal are to define the spectrum of MsgC fragments that are recognized by human serum in health and PcP disease, and to identify individual antigenic epitopes found on disease relevant Msgs. We will determine if these epitopes are unique or cross-reactive with other variants of Msg, and will define the hierarchy of epitopes seen by individual patients. We will also study the frequency of immune recognition of the individual epitopes in different patient populations. We propose to identify the spectrum of MsgC fragments recognized by human serum by generating a phage display library expressing a diversity of MsgC fragments, and will use positive and negative phage selection to identify disease relevant MsgC fragments. These fragments will be studied for individual epitopes using a single chain variable region (scFv) antibody phage library. This library will be a set of monoclonal probes of human origin useful for identifying individual epitopes of MsgC. These newly identified epitopes will, in the long term, be used to probe immune reactivity in HIV+ patients undergoing HAART.