Shanjana Awasthi

Efficacy of Antigen 2/Proline-Rich Antigen cDNA-Transfected Dendritic Cells in Immunization of Mice against Coccidioides posadasii

Coccidioides posadasii causes coccidioidomycosis, or Valley fever, in the endemic regions of the Southwestern United States. The susceptibility to C. posadasii infection has been attributed to a decreased Th1 cellular response. APCs, especially dendritic cells (DCs), play an important role in the activation of Th1 response. In this study, we investigated the efficacy of a DC-based vaccine against C. posadasii in a mouse model of coccidioidomycosis. We intranasally immunized C57BL6 mice with syngeneic, bone marrow-derived DCs (JAWS II cells) transfected with a cDNA encoding the protective Coccidioides-Ag2/proline-rich Ag. The immunized mice were lethally challenged with C. posadasii through either an i.p. or intranasal route. Upon necropsy after 10 days of infection, fungal burden in lung and spleen of immunized mice was significantly reduced as compared with the control animals. The lung tissue homogenates of immunized animals showed higher levels of IFN-γ. Histologically, lung tissues of immunized mice were in better condition than the control mice. To further investigate, we studied the biodistribution and trafficking of injected DCs by nuclear imaging techniques. For this purpose, the transfected DCs were radiolabeled with 111In-oxime. Scintigraphic images showed that most of the label remained in the gastrointestinal tract. A significant amount was also observed in lung, but there were negligible circulating 111In label in blood. The results suggest that the DCs have a potent immunostimulatory activity, and immunization with DCs transfected with Ag2/proline-rich Ag-cDNA induces protective immunity against C. posadasii in C57BL6 mice.

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D

BackgroundCoccidioidomycosis or Valley Fever is caused by Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known.MethodsIn this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.ResultsWe found that in lavage fluid samples of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.ConclusionOur results suggest that the C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.

Susceptibility of TLR4-defective C3H/HeJ mice to Coccidioides posadasii infection

Coccidioides posadasii is one of the two fungal pathogens that cause coccidioidomycosis. The inhalation of air-borne arthroconidia leads to the formation of endospore-forming spherules in the lungs and pulmonary infection. In severe condition, the endospores are disseminated to other non-pulmonary organs in the body. The Toll-like receptors (TLR) expressed by a number of immune and non-immune cells can significantly impact the host defense and susceptibility to C. posadasii infection. In this study, we infected TLR4-defective C3H/HeJ mice with a sublethal dose of C. posadasii and studied fungal dissemination, mortality and humoral response. We also measured IL-12 cytokine secreted by C. posadasii-infected dendritic cells. We found that the C3H/HeJ mice were equally susceptible to C. posadasii as compared to C3H/OuJ mice which have intact TLR4. No significant changes were observed in pulmonary fungal load, survival and humoral response. The blockade of TLR4 did not affect C. posadasii-induced IL-12 secretion. However, the fungal counts were 10 times less in spleens of C3H/HeJ mice as compared to C3H/OuJ mice (P<0.05). Our results suggest that the TLR4 may not be involved in inducing protective host defense against C. posadasii, but it appears to be critical for fungal dissemination.