Masters Thesis

Pulmonary Aspergillosis in Mice and Chickens

The fungus Aspergillus can cause a serious pulmonary infection referred to as Invasive Pulmonary Aspergillosis, (IPA) in immunocompromised patients, such as neutropenic patients, with 80% mortality if untreated. Aspergillosis also affects the poultry industry as chickens and turkeys are highly susceptible, leading to meat condemnation. Given the limited number of approved drug treatments to treat IPA, we worked with Molecular Express Inc. to develop a liposomal vaccine with Aspergillus proteins to prevent aspergillosis in susceptible humans and poultry. The VesiVax® liposomal vaccines include Aspf3 and Aspf9 proteins and lipidated tucaersol adjuvant (LT1) LAsV-LT1. The Aspf3and Aspf9 vaccines were administered subcutaneously (SC) and intranasally (IN) to outbred female Swiss-Webster (SW) mice or subcutaneously (SC) and mucosally (MU) to male and female SPF chickens. To do these studies, we first had to establish a neutropenic model of IPA in SW female mice using cyclophosphamide. The immunosuppressed mice were challenged with A. fumigatus ATCC #13073 and then monitored for morbidity and survival. Having established the model, it was used to test the efficacy of the mixture of LAsV-LT1 vaccines. Groups of mice were vaccinated with Aspf3 and Aspf9 LAsV-LT1 vaccines on d0, d21/d23 and d42/44 and spleens and blood collected on d45. Mice were then immunosuppressed with cyclophosphamide, challenged with A. fumigatus and some groups treated 12h post-challenge with 7.5 mg/kg IV AmBisome® (liposomal amphotericin B, AmBi), 40 mg/kg oral Vfend® (voriconazole, Vori) or Phosphate buffered saline (PBS). Serum and lungs were collected post challenge to analyze fungal burden and anti-spore antibody titers in the serum. Remaining mice were monitored for morbidity for 21 days post challenge. The LAsV vaccines protected the neutropenic, SW female mice against infection with A. fumigatus and this protection was increased with the use of delayed antifungal drug treatment, AmBi or Vori, as measured by survival and fungal burden. Anti-Aspf3 and anti-Aspf9 IgG1 and TL-4 secreting splenocytes showed that the LAsV-LT1 vaccines elicited a Th2 response and this protection correlated with an increased anti-spore antibody titer in the serum of vaccinated mice. Since there was no previously available model of chicken IPA, a broiler chicken IPA model had to be first set up in dexamethasone immunosuppressed male and female SPF chickens for subsequent testing of the LAsV-LT1 vaccines. Broiler chickens were immunosuppressed with different concentrations of Dexamethasone Sodium Phosphate (DSP) and challenged with A. fumigatus ATCC #13073 (8 X 10^8) to determine the most effective immunosuppressive dose and fungal inoculum. Chickens were monitored for disease scores post challenge and lungs and tracheas were collected 4 days post challenge for fungal burden. BID intramuscular dexamethasone dosing (8 mg/kg) on the day before, on the day of fungal challenge and for 3 more days after fungal challenge produced the most severe IPA and was used for the vaccination studies. The vaccination route (SC or MU) and days of vaccination were then varied to determine the most protective route and regimen. Chickens were monitored for disease scores post challenge and lungs, tracheas, and blood collected 4 days post challenge for determination of fungal burden and serum anti-spore antibody titers. In the SPF chickens, the LAsV-LT1 vaccines provided protection based on decreased diseases scores and decreased tissue fungal burden. The LAsV-LT1 vaccines still provided protection when the route of delivery was modified to just MU administration and when the number of MU doses was decreased from three to two. The increase in the serum anti-spore antibody titers in vaccinated chickens was similar to the increase in serum anti-spore antibody titers seen in the vaccinated mice. Given the increased survival, decreased fungal burden in the lungs, and increased serum anti-spore antibody titers in both mice and chickens, the LAsV-LT1 vaccines appear to be effective in protecting both mammalian and avian species against IPA and offer a potentially less expensive, and less labor-intensive treatment for this serious infection

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