Invasive fungal infections cause millions of deaths each year and they are refractory to treatment. Patients at the greatest risk of developing life-threatening fungal infections have weakened immune systems or various other lung disorders. Aspergillus fumigatus and related fungal species cause pulmonary invasive aspergillosis. Even after treatment with antifungals such as amphotericin B (AmB) packaged in liposomes, one-year survival among immunocompromised patients with invasive aspergillosis is only 25 to 60%. Antifungals are toxic to patients with numerous side effects and no current treatment clears the lungs of fungi. Without a doubt, there is room for improving antifungal therapeutics to treat aspergillosis. Immunoliposomes are liposomes coated with a monoclonal antibody (mAb) or other protein with specificity for a surface antigen on target cells, enabling the active delivery of a therapeutic drug to a specific cell type. Drug loaded immunoliposomes greatly improve cell-type specificity and reduce cytotoxicity such that higher doses of drug may be delivered to target cells than with liposomes. The overarching goal of this exploratory proposal is to demonstrate that fungal-cell wall targeted immunoliposomes loaded with antifungal agents have increased efficacy over drug loaded liposomes. Our experimental hypothesis is that AmB loaded immunoliposomes coated with monoclonal antibodies or Dectin-1 targeting A. fumigatus cell wall antigens will show enhanced fungal-specific delivery and antifungal activity over untargeted liposomes. Two innovations further our goal. First, we isolated a battery of A. fumigatus cell-wall specific mAbs that bind differentially to various regions on hyphae, germ tubes and conidia, that will enable targeted delivery of immunoliposomes to fungi in vitro and in vivo. Second, we furthered the development of simplified protocols for constructing small batches of fluorescent AmB immunoliposomes and screening methods that will accelerate our examining various targeted-liposomal formulations to fungi in vitro and in vivo in the lungs of infected mice. Our first specific aim is to screen among mAbs and Dectin-1 for targeting protein constructs that achieve the most efficient uptake of fluorescent AmB immunoliposomes into A. fumigatus cells. Results will be quantified by fluorescent microscopy, ELISA, and by assaying fungal cell survival. Our second specific aim is to show that treating infected mice with AmB loaded immunoliposomes targeting A. fumigatus cell wall component(s) kills more fungal cells and increases mouse survival rates relative to untargeted drug loaded liposomes. Results will be evaluated by examining the (1) reduction in A. fumigatus viable cell numbers, (2) alteration in lung pathology, and (3) improvement in mouse survival rates. A team of well acquainted scientists with expertise in immunology, immunoliposomes, fungal biology and pathology, and biochemistry has been assembled.
/ Public Health Relevance Invasive fungal infections cause millions of deaths each year and the are refractory to antifungal drug treatment, such that among immunocompromised patients the one-year survival rate after treatment can be as low as 25%. Our goal is to demonstrate that antifungal drug loaded immunoliposomes targeting pathogenic fungal cell wall components improve treatment efficacy in mouse models. We will use amphotericin B loaded liposomes coated with our own monoclonal antibodies or Dectin-1 targeting Aspergillus fumigatus cell wall antigens and show their enhanced antifungal activity and reduced toxicity over untargeted drug loaded liposomes.
