Healthy Human T-Cell Responses to Aspergillus fumigatus Antigens

Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H)1-T(H)17) and destructive allergic (T(H)2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown.To determine whether Asp f proteins are strictly associated with T(H)2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H)1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals.Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases

Prophylactic and Therapeutic Potential of Asp f1 Epitopes in Naïve and Sensitized BALB/c Mice

Background: The present study examines a hypothesis that short allergen-derived peptides may shift an Aspergillus fumigatus (Afu-) specific TH2 response towards a protective TH1. Five overlapping peptides (P1-P5) derived from Asp f1, a major allergen/antigen of Afu, were evaluated for prophylactic or therapeutic efficacy in BALB/c mice. Methods: To evaluate the prophylactic efficacy, peptides were intranasally administered to naïve mice and challenged with Afu-allergens/antigens. For evaluation of therapeutic efficacy, the mice were sensitized with Afu-allergens/antigens followed by intranasal administration of peptides. The groups were compared for the levels of Afu-specific antibodies in sera and splenic cytokines evaluated by ELISA. Eosinophil peroxidase activity was examined in the lung cell suspensions and lung inflammation was assessed by histopathogy. Results; Peptides P1-, P2- and P3 decreased Afu-specific IgE (84.5~98.9%) and IgG antibodies (45.7~71.6%) in comparison with Afu-sensitized mice prophylactically. P1- and P2-treated ABPA mice showed decline in Afu-specific IgE (76.4~88%) and IgG antibodies (15~54%). Increased IgG2a/IgG1 and IFN-γ/IL-4 ratios were observed. P1-P3 prophylactically and P1 therapeutically decreased IL-5 levels and eosinophil peroxidase activity. P1 decreased inflammatory cells' infiltration in lung tissue comparable to non-challenged control. Conclusion: Asp f1-derived peptide P1, prophylactically and therapeutically administered to Balb/c mice, is effective in regulating allergic response to allergens/antigens of Afu, and may be explored for immunotherapy of allergic aspergillosis in humans

<i>A. fumigatus</i> antigens recognition by 20 healthy donors.

<p>(a) IFN-γ (b) IL-4 and (c) IL-17 responses. Blood was collected from 20 healthy donors and triplicate samples of 1×10⁵ PBMCs were stimulated with medium, 5 µg/ml PHA or 1 µg/ml CHE or recombinant <i>A. fumigatus</i> antigens for 24–48 hr at 37°C/5%CO₂. The mean antigen-specific spot forming cells, SFC (after background subtraction of control wells with no antigen) is plotted.</p

Asp f proteins.

*<p>Asp f1 cDNA could not be cloned downstream of the pTrc promoter in pTrcHis2.</p><p>#Molecular sizes in bold correspond to the full length protein.</p><p>Proteins in bold were successfully expressed in <i>E. coli</i>.</p

IFN-γ ELISPOT assay with purified CD4⁺ and CD8⁺ T-cells.

<p>CD4⁺ or CD8⁺ T-cells (1×10⁵) were isolated from strong responders' PBMC, incubated with 1 µg/ml of (a) rAsp f3 and (b) rAsp f9 with autologous irradiated PBMC (5×10⁴) in triplicates for 24 hr at 37°C/5% CO₂. PHA (5 µg/ml) was used as a positive control. The mean antigen-specific spot forming cells, SFC (after background subtraction of control wells with no antigen) ± SD is shown.</p

IFN-γ stability assay with rAsp f3 and rAsp f9 after period of 6 months.

<p>To determine whether the IFN-γ responses are stable over time, blood was collected from high responders after six month interval of previous assay and triplicate samples of 1×10⁵ IFN-γ PBMC were stimulated with medium and 1 µg/ml rAsp f3 (a) or rAsp f9 (b) for 18–24 hrs at 37°C/5%CO₂. The mean antigen-specific spot forming cells, SFC (after background subtraction of control wells with no antigen) ± SD is shown. The figures show one representative data from three independent experiments.</p

IFN-γ, IL-4 and IL-17 T-cell responses of healthy donors (n = 20) to CHE and recombinant antigens of <i>A. fumigatus</i>.

<p>Strong responders (SR) were defined ≥20 SFU/10⁵ cells, weak responders <20 SFU/10⁵ cells. NR  =  no SFU quantified. Data are derived from means of triplicate measurements in three independent experiments.</p

Murine models of Aspergillosis: Role of collectins in host defense

691-700Aspergillus fumigatus, a ubiquitous fungus, causes a wide spectrum of clinical conditions ranging from allergic to invasive aspergillosis depending upon the hosts’ immune status. Several animal models have been generated to mimic the human clinical conditions in allergic and invasive aspergillosis. The onset, duration and severity of the disease developed in models varied depending on the animal strain/fungal isolate, quantity and mode of administration of fungal antigens/spores, duration of the treatment, and type of immunosuppressive agent used. These models provide insight into host and pathogen factors and prove to be useful for evaluation of diagnostic markers and effective therapies. A series of studies established the protective role of collectins in murine models of Allergic Bronchopulmonary Aspergillosis and Invasive Pulmonary Aspergillosis. Collectins, namely surfactant protein A (SP-A), surfactant protein D (SP-D) and mannan binding lectin (MBL), are pattern recognition molecules regulating both innate and adaptive immune response against pathogens. In the present review, we discussed various murine models of allergic and invasive aspergillosis and the role of collectins in host defense against aspergillosis