Pneumocystis (PC) pneumonia remains a serious complication of HIV infection and other immunocompromised states. Recent ICD9/10 data show that since 2008 there are consistently 14-15,000 hospitalizations per year with an average cost of close to $1B per annum in the US alone. Moreover treatment for PCP has not changed in 25 years and there is concern of anti-microbial resistance and drug:drug interactions with TMP-SMX (the frontline therapy for PCP). The well-known inverse relationship between CD4+ lymphocyte count and the risk of PC infection does not hold all of the answers to mechanisms of host defense against this infection. In the prior funding period, we made significant progress on defining the Pneumocystis transcriptome in both ascus and trophozoite forms as well as the surface proteome of both forms through surface labeling of the fungus using techniques that were developed for Candida. We identified troph proteins such as Meu10, a GPI-anchored protein, as well as ascus specified proteins such as glucan synthetase 1 (GSC1). We were able to clone and express the GSC1 ectodomain in yeast, and immunization of mice with GSC1 resulted in antibodies that stain the surface of the ascus and prevent transmission of Pneumocystis in cohousing experiments. We also made significant progress on diagnostic assays to discriminate colonization from infection. Several groups ae now using quantitative PCR with copy number thresholds, which may be a valid approach. In the prior funding period developed and validated a troph specific assay that selectively eradicates the ascus. Furthermore, RNAseq analysis revealed the troph to be much more metabolically active than the ascus. Thus, we have developed life- form specific assays that will be tested in clinical specimens. We hypothesize that GSC1 immunization can prevent pneumocystis transmission and that mucosal immunization is superior to subcutaneous immunization. We also predict that GSC1 antisera or monoclonal antibodies directed against the GSC1 ectodomain can mitigate Pneumocystis IRIS. Moreover, as our RNAseq analysis has also revealed that the troph is clearly the replicative form of the organism in the lung, we hypothesize that an assay to detect troph specific genes will be diagnostic of PJP due to the detection of the replicative form of the organism. We will also determine the genetic heterogeneity of these target genes in samples from 4 continents. We will test this hypothesis with the following specific aims: Aim 1. Determine the immunogenicity and efficacy of systemic (subcutaneous or IM) versus mucosal GSC1 immunization to prevent PCP. Aim 2. Determine the efficacy of anti-ascus or anti-troph antibodies to treat established PCP and immune reconstitution syndrome (IRIS). Aim 3. Determine and validate life-form specific PCR/CRISPR assays in murine and human samples and assess genetic heterogeneity of these targets in samples from North America, Africa, and western and eastern Asia.