YESHIVA UNIVERSITY
Grant: $52,735 - National Institutes of Health - Jun. 4, 2009
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Award Description: The dimorphic fungus Histoplasma capsulatum var. capsulatum [Hc] is the most prevalent cause of fungal respiratory infections. Hc infects approximately 500,000 individuals in the USA each year. Infection usually results in a mild, often asymptomatic respiratory illness, but may progress to life-threatening systemic disease, particularly in individuals with AIDS. Historically, immunity to Hc has been considered to be dependent on cell-mediated immune responses. Individuals with impaired cellular immunity, particularly patients with AIDS, are more likely to have disseminated histoplasmosis. New therapies are urgently needed for treatment of fungal diseases. Currently available medications for systemic fungal infections often fail to work in the setting of impaired immunity, and many AIDS patients require life-long prophylaxis to prevent recurrent disease. I have generated three mouse monoclonal antibodies (mAbs) to the surface of Hc cells that were derived from splenocytes of mice immunized with Hc yeast that significantly prolong survival in a murine histoplasmosis model (131). This is the first time that antibody has been reported to affect the course of experimental Hc infection. The broad objective of this proposal is to gain insight into the role of antibody in histoplasmosis: to determine the mAbs mechanism of action and to characterize the antigen that the mAbs recognize. Additionally, since this antigen can elicit protective antibodies it is a potential vaccine candidate. The specific Aims are: 1. To map the epitope of mAb 9C7 and generate additional mAbs to Hc; 2. To investigate the mechanism(s) of antibody-mediated protection against Hc in vivo; 3. To investigate the mechanism(s) of antibody-mediated protection against Hc in vitro; 4. To determine the efficacy of the histone H2B and the mAb 9C7 epitope as vaccines.
Project Description: Progress related to prior Aim 1. We generated fragments of the Hc H2B expressed in bacteria and initially localized the epitope of mAb 9C7 to the first half of the protein. Subsequent fragments were serially produced in E. coli enabling the localization of the epitope for the mAbs to H2B h to a 14 amino acid peptide at amino acids 36-52. Additional mapping with alanine substitution has revealed that the first half of the protein is required for binding of the mAb. New mAbs are being produced to Hc targets. Progress related to prior Aims 2 & 3. The key findings are that the protective mAb results in increased phagosome maturation, greater phagosome-lysosome fusion, and a greater reduction in phagosomal pH. These alterations are consistent with the increased capacity of the infected macrophage to inhibit fungal growth and even result in fungal killing. Over the past year, we have further shown by subcellular fractionation of macrophage that maturation of the phagosome occurs and there is increased antigen processing. In turn, we have found that T cell activation is augmented when the yeast cells are opsonized by protective mAb. Furthermore, we have determined that antibody isotype impacts efficacy. Progress related to prior Aim 4. Pilot studies suggested that immunizations using H2B with Freund?s adjuvant were protective. However, when large numbers of mice were subjected to this immunization approach, we found that it did not induce a statistically significant protective immune response to murine histoplasmosis. We were able to obtain CpG for use as an adjuvant. Our recent data shows that CpG with H2B induces protection. Notably, CpG alone also provides statistically improved protection, but the protection is less than the combination of CpG and H2B.
Jobs Summary: na (Total jobs reported: 0)
Project Status: Less Than 50% Completed
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