Culicoides allergens expressed in insect cells induce sulphidoleukotriene release from peripheral blood leukocytes of horses affected with insect bite hypersensitivity

Abstract

Introduction: Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis in horses caused by bites of Culicoides spp. The allergens are salivary gland proteins from these insects, and nine major allergens from Culicoides obsoletus have been identified and expressed in E. coli. However, proteins expressed in procaryotic systems have limitations in cellular assays, particularly in functional assays assessing the allergen-induced release of mediators in vitro, such as sulphidoleukotrienes (sLT) from basophils. The aims of the study were to produce functional Culicoides allergens in insect cells, to assess their allergenicity using a sLT release assay, and to relate the sLT release with IgE sensitization to the respective allergens using ELISA.

Methods: Eight major Culicoides obsoletus allergens (Cul o 1P, Cul o 2P, Cul o 3, Cul o 5, Cul o 7, Cul o 8, Cul o 9, and Cul o 11) were expressed in insect cells and purified. sLT release from peripheral blood leukocytes (PBL) following stimulation with the eight Culicoides allergens was measured in 28 IBH-affected and 24 healthy control horses. Allergen-specific serum IgE levels was determined by ELISA.

Results: The eight major allergens were successfully expressed in insect cells and purified. All allergens induced a significantly higher sLT release from PBL of IBH-affected horses compared to healthy controls. There was a high correlation and substantial to excellent agreement between sLT release and serum IgE levels for six Culicoides allergens, while for two, the agreement was moderate. Positivity rates in IBH horses were usually higher in IgE serology, but more false-positive results were obtained. The allergens performing best in both assays were Cul o 3, Cul o 8, and Cul o 9, with very high specificity and good sensitivity.

Discussion: Insect-cell-expressed Culicoides recombinant allergens are functionally relevant and will open new opportunities for the study of Culicoides hypersensitivity not only in horses but also potentially in human patients or other species. They will also greatly improve IBH diagnostics using cellular assays and IgE serology.

Keywords: Culicoides allergens; horse; insect bite hypersensitivity; insect cell expressed recombinant proteins; serum IgE; sulphidoleukotriene release.

Figure 1

Released sulphidoleukotriene (sLT) following stimulation of peripheral blood leukocytes with C. obsoletus (CO) and C. nubeculosus (CN) whole body extract. (A) Concentration of sLT in healthy (H) and IBH-affected (IBH) horses shown as median (─) for each group. Dots represent the value of horses. Mann–Whitney U test was used to compare the difference between the groups, ****p ≤ 0.0001. (B) Concentration of sLT in the IBH-affected horses following stimulation with CN and CO extracts. Dots connected with a line represent the values from a single horse. Comparison between extracts was performed with Wilcoxon test, *p ≤ 0.05.

Figure 2

SDS-PAGE of purified and dialyzed Culicoides allergens expressed in High Five insect cells. (A) Protein staining: 2 µg of each allergen was loaded on the gel. (B) Immunoblot: 0.5 µg of each allergen was loaded on the gel. M: PageRuler marker, 1: Bac-Cul o 1P, 2: Bac-Cul o 2P, 3: Bac-Cul o 3, 4: Bac-Cul o 5, 5: Bac-Cul o 7, 6: Bac-Cul o 8, 7: Bac-Cul o 9, 8: Bac-Cul o 11.

Figure 3

Titration of a pool of insect-cell-expressed (Bac-) C. obsoletus allergens in cellular antigen stimulation test. Released sulphidoleukotriene (sLT) following stimulation of peripheral blood leukocytes with different concentration of the pool of Bac-Cul o 1P, Cul o 2P, and Cul o 8. The sLT is shown for each concentration as median with interquartile range (IQR). The titration was done with PBL from eight IBH-affected horses at the beginning of the study.

Figure 4

Released sulphidoleukotriene (sLT) following stimulation of peripheral blood leukocytes with the eight insect-cell-expressed (Bac-) C. obsoletus allergens. Concentration of sLT plotted for healthy (H) and IBH-affected (IBH) horses shown with median for each group in red. Each dot represents the value of single horse. Mann–Whitney U test was used to compare the difference between the groups, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 5

Serum IgE from healthy (H) and IBH-affected (IBH) horses specific for the eight insect-cell-expressed (Bac-) C. obsoletus allergens tested by ELISA. OD405 value of each horse plotted and shown with the median for the group in red. Mann–Whitney U test was used to compare the difference between the groups, ****p ≤ 0.0001.

Figure 6

Percentage of horses positive in CAST and IgE ELISA on the eight insect-cell expressed Culicoides obsoletus allergens. Percentage of horses positive in CAST (white columns) and in IgE ELISA (gray). Data from healthy horses shown as columns with patterns.

Figure 7

Agreement between CAST and IgE positivity. Agreement between CAST and IgE positivity for the eight Culicoides obsoletus allergens. (A) H control horses. (B) IBH-affected horses. The red and orange colors show full agreement (CAST+/IgE+ and CAST−/IgE−, respectively). Green, CAST−/IgE+; blue, CAST+/IgE−.

Figure 8

Cumulative positivity to the eight C. obsoletus recombinant allergens in IgE serology (A) and CAST (B). Percentages of IBH-affected and healthy horses against the number of recombinant allergens to which they react.