The Characterization of Pneumocystis Surface Antigens

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Abstract

We have an ongoing project to characterize the antigens of mouse, rat, and human Pneumocystis. We have previously purified the major surface glycoprotein (MSG) of both rat and human pneumocystis using HPLC. It is necessary to use Pneumocystis from both sources because the organisms are different species. Subsequently, we identified a number of clones from a cDNA library of rat Pneumocystis that contain genes encoding for the MSG. These clones are clearly related but not identical, demonstrating that multiple genes encode the MSG. We have continued studies to characterize potential antigens of Pneumocystis. We have cloned a number of human Pneumocystis MSG genes and have expressed a full-length MSG in two fragments. We developed an ELISA to examine antibody responses to these antigens, and have utilized it to examine sera from patients with or without HIV infection, and with or without a history of PCP, as well as sera from a variety of healthy conrols. In about 15% of healthy patients followed serially we have been able to document changes in antibody titers, suggesting that these individuals have developed reinfection or reactivation of Pneumocystis infection. We will continue these studies to better understand the epidemiology of Pneumocystis infection in humans. We have also identified the unique expression site of MSG in human Pneumocystis, and can now identify the MSG variants that are expressed in a patient with PCP. Within this expression site we have identified a region of tandem repeats that varies among different Pneumocystis isolates, and thus provides a new method for typing human Pneumocystis. We have sequenced MSG variants from a single human pneumocystis isolate and have shown that the isolates FM1p, into 2 subgroups that are genetically distinct. These results led to studies that demonstrated that the MSG repertoire is very diverse, while the MSG repertoire in rat and mouse Pneumocystis is identical or very limited among different isolates. We are currently evaluating the utility of RFLP evaluations of MSG genes as a typing method for human Pneumocystis. We are also studying a protein related to kexin, a protease of yeast, that appears to be an antigen of rodent pneumocystis. We have cloned and expressed recombinant kexin from human and mouse pneumocystis and are currently examining immune responses to the recombinant protein. The goal of this study is to better understand the pathogenesis of Pneumocystis pneumonia with the hope that we can use this information to control or prevent this disease.