Purpose of Study: Aspergillus infection is a major cause of mortality in the severely immunocompromised and vaccine development is a high priority. Heat shock proteins (HSPs) are carriers of immunogenic peptides that potently stimulate dendritic cell (DC) activation and antigen-specific immunity. Asp f2 is an abundantly produced A. fumigatus antigen. Here, we characterized DC activation and antibody responses to HSP110/Asp f2 complex. Methods: DC activation: A HSP110/Asp f2 complex was generated. Bone marrow derived DCs from wildtype and TLR 4 deficient mice were generated. DCs were stimulated under the following conditions: 1) vehicle; 2) HSP 110; 3) Asp f2; 4) HSP110/Asp f2 complex; or 5) LPS (positive control). Expression of CD86, a key co-stimulatory molecule required for antigendriven T-cell activation was quantitated by flow cytometry. IgG subclasses: C57BL/6 mice were sensitized IP on days 0 and 14 with vehicle; HSP 110; Asp f2; or HSP110/Asp f2 complex. Asp f2-specific IgG responses were determined by ELISA on days 14 and 25 after challenge. Results: DC activation: HSP110 increased CD86 surface expression in wildtype DCs compared to unstimulated cells. In contrast, no increase in CD86 expression occurred in TLR 4-/- DCs after HSP110 stimulation, indicating that HSP110-mediated stimulation of CD86 expression is TLR 4-dependent. Asp f2 alone was a more potent stimulus of CD86 expression than HSP110 in wildtype DCs and the HSP110/Asp f2 complex had comparable to increased stimulation of CD86 expression compared to Asp f2 alone. In TLR 4-/- DCs, Asp f2 stimulated CD86 expression to a similar degree as in wildtype DCs. Asp f2 alone and complexed to HSP110 generated similar levels of CD86 expression in TLR 4-/- DCs. IgG subclasses: HSP110/Asp f2 complex was immunogenic in vivo. Sensitization with the complex elicited an approximately 10-fold increase in Asp f2-specific IgG2A serum levels on day 25 compared to sensitization with Asp f2 alone. IgG1 responses were similar between mice sensitized with HSP110/Asp f2 and Aspf f2 alone. Conclusions: HSP110-mediated stimulation of CD86 expression on DCs is TLR-4-dependent whereas Asp f2 is able to stimulate CD86 expression independently of TLR-4. The ability of major recombinant Aspergillus antigens to induce DC activation merits further study. In vivo, sensitization with the HSP 110/Asp f2 complex induced an increased Asp f2-specific IgG2A response compared to sensitization with Asp f2 alone. IgG2A responses are induced by IFN-γ 61472;and suppressed by IL-4. Thus, an increase in antigen-specific IgG2a responses reflects skewing of T-cells to the type I phenotype. The ability of HSP110 to affect both innate and acquired immunity makes it a promising strategy for immunotherapy against Aspergillus infection.
Full conference title:
15th Annual Focus on Fungal Infections
- FFI 15th (2005)