Modulation of host immune defense by Pneumocystis beta-glucans Pneumocystis are fungal organisms associated with the development of pneumonia in immunocompromised hosts. When Pneumocystis pneumonia (PcP) develops it carries great morbidity and mortality even upon administration of appropriate treatment. In the immunosuppressed patient, PcP can only be prevented with antibiotic prophylaxis. Unfortunately, most of the cases occur in patients in which prophylaxis was never instituted. The main reason for this lies in the lack of consensus guidelines regarding when to initiate prophylaxis in patients at risk. Specifically, the duration of therapy, fear of secondary effects from the preventive drugs, drug allergies and patient compliance have all contributed to the failure of adequate prophylactic treatments. A vaccine that can be administered early to patients at risk of PcP is therefore desperately needed. Beta-glucans (BG) are carbohydrates found in the cell wall of fungi (including Pneumocystis) which modulate both the innate and adaptive immune systems and are potentially excellent agents to use as vaccine adjuvants. Vaccine adjuvants with good immunogenic properties are essential for effective vaccine therapies especially when using recombinant antigens which are generally poorly immunogenic. As part of their innate activation BG induce stimulation of dendritic cell (DCs) and subsequent CD4 differentiation. BG have been additionally shown to modulate B cell responses independent of CD4 cells. All these properties make BG an excellent tool for the manipulation of CD4-dependent and CD4-independent immune responses. The ability of modulating immune responses in the absence of CD4 cells is of particular importance. Specifically as PcP mostly affects patients with low CD4 counts such as with HIV, individuals with autoimmune diseases and hematological malignancies as a result of the diseases themselves or the treatments received. Hence, understanding how Pneumocystis-derived BG (PCBG) modulate the immune responses will allow us to develop tools to manipulate the innate and adaptive host immune response to better fight this and potentially other fungal infections. Therefore, the objectives of this application are to 1. dissect the mechanism(s) by which PCBG activate B cells and the participation of DC in this process, to better understand CD4-independent mechanism of immune response against fungal components and 2. evaluate the effect of PCBG as a vaccine adjuvant together with Pneumocystis antigens in a CD4-replete and CD4-depleted murine models of Pneumocystis infection. We anticipate that the results of these investigations will enhance the understanding of PCBG activation of CD4-independent mechanisms of fungal protection and result in the identification of new targets for therapeutic exploitation in the treatment and prevention of PcP. In addition, we expect to develop a mouse model of Pneumocystis vaccination that could potentially be translated to human use. If PCBG proves it potential role as a potent vaccine adjuvant it could also be applied to other fungal, bacterial and viral vaccines, to enhance protection. The career development plan proposed here will help me gain the knowledge and expertise in B cell responses to PCBG to complement my prior training on human peripheral DC. Additionally, I will gain expertise in mouse model manipulation to achieve the objectives of this application. The future experiments outlined here will also enable me to move into the field of plasma membrane microdomains and initiate an investigation into individualized responses to vaccination through the latest high throughput next generation sequencing technologies. Ultimately, the career development and research plans outlined here will provide me with the skills needed to achieve my long term career goal which is to become an independent investigator with expertise in host defense against fungal infection particularly in the fields of non-CD4 responses and cell- mediated immunotherapeutics.

Public Health Relevance

Patients with defective immune systems (particularly those defective in their CD4 T cell populations) are at risk of death from Pneumocystis pneumonia (PcP) even upon administration of appropriate treatment. PcP can be prevented with antibiotics, but preventive treatments are often not prescribed due to difficulties recognizing when to initiate prevention, duration of treatment, drug allergies and secondary effects. Patient compliance required during the extended periods of antibiotic prophylaxis also confounds effective prevention. Therefore, a protective vaccine against PcP is desperately needed to prevent this infection. The beta-glucan cell wall components of Pneumocystis organisms (PCBG) are potent stimulators of our immune system and potentially great targets for vaccine adjuvants. Vaccines using extracted Pneumocystis molecules generally lose this portion of the organism upon preparation, limiting their potential. Herein, we propose to use beta-glucan from Pneumocystis to develop a Pneumocystis vaccine in a murine model of infection. In addition, we will further investigate the molecular mechanisms involved in PCBG activation of B cell and the role of dendritic cells modulating this process in the absence of CD4 cells. We believe that our proposed studies will enhance the understanding of PCBG modulation of CD4-independent protective responses and potentially identify new targets for drug therapy. We strongly believe that the proposed research has a very high relevance for public health as these studies can also help in development of treatments for other fungal pneumonias.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Clinical Investigator Award (CIA) (K08)
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Special Emphasis Panel (ZHL1)
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Colombini-Hatch, Sandra
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Mayo Clinic, Rochester
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Ali, Mohamed F; Dasari, Harika; Van Keulen, Virginia P et al. (2017) Canonical Stimulation of the NLRP3 Inflammasome by Fungal Antigens Links Innate and Adaptive B-Lymphocyte Responses by Modulating IL-1? and IgM Production. Front Immunol 8:1504
Ali, Mohamed F; Dasari, Harika; Van Keulen, Virginia P et al. (2017) Microbial Antigens Stimulate Metalloprotease-7 Secretion in Human B-Lymphocytes Using mTOR-Dependent and Independent Pathways. Sci Rep 7:3869
Calero-Bernal, Maria L; Martin-Garrido, Isabel; Donazar-Ezcurra, Mikel et al. (2016) Intermittent Courses of Corticosteroids Also Present a Risk for Pneumocystis Pneumonia in Non-HIV Patients. Can Respir J 2016:2464791
Ali, Mohamed F; Driscoll, Christopher B; Walters, Paula R et al. (2015) ?-Glucan-Activated Human B Lymphocytes Participate in Innate Immune Responses by Releasing Proinflammatory Cytokines and Stimulating Neutrophil Chemotaxis. J Immunol 195:5318-26