Analysis of the immune responses of horses with insect bite hypersensitivity to recombinant salivary gland proteins of Culicoides spp

Background:
Insect Bite Hypersensitivity (IBH) of horses is an IgE mediated allergy to salivary proteins of Culicoides spp. (biting midges). IBH occurs throughout the world, affecting 3% of horses and donkeys in the UK but up to 60% of susceptible horses under some circumstances. Intra-dermal exposure of allergic horses to Culicoides antigens elicits an acute phase response with local vasodilation, oedema and pruritis, followed by a late phase reaction characterised by infiltrating T-cells, mast cells and eosinophils. Allergic horses have Culicoides antigen specific IgE antibodies and antigen reactive T-cells which secrete more IL-4 but less Interferon gamma or regulatory cytokines (IL-10 and TGF-β) than T-cells of healthy controls. The long term aim of our research is to develop an effective immuno-therapy to induce tolerance; an essential prerequisite of such a therapy is to have an available source of purified antigen. We have constructed three cDNA libraries: one using salivary gland mRNA isolated from a single laboratory bred species (C. nubeculosus ) and two from the pooled salivary glands of wild caught insects of the Pulicaris and Obsoletus taxonomic groups, which represent the commonest species found biting horses in the UK and Europe. From these we have identified cDNAs encoding the most abundant proteins in Culicoides saliva.

Hypothesis:
Prior studies have identified several putative allergens among Culicoides salivary gland proteins. We propose that these same proteins expressed in insect cell cultures by recombinant Baculovirus will retain their IgE binding and stimulate a T-helper 2 type response in allergic horses confirming their status as major allergens.

Aims:
To construct recombinant Baculovirus which express between 10 and 20 of the major salivary gland proteins found in Culicoides saliva.
To test the ability of the recombinant proteins to bind IgE antibodies from serum of allergic horses.
To compare, in vitro, the proliferation and cytokine production of T-cells from healthy and allergic horses in response to recombinant Culicoides proteins.

CASE partner: to be confirmed