IgE is the molecular form of antibodies that when bound to mast cells will initiate allergic (hypersensitivity type I) reactions upon encounter with the allergen against which it is specific. Little is known about the molecular nature of antibody recognition of individual allergens. This project aims at defining the characteristics of human IgE recognition of allergens and to provide a resource of human antibodies targeting such allergens.
To further the understanding of allergic reactions we have sequenced genes encoding human gE as it is found in allergic individuals (i.e. in allergy-encoding transcriptomes). We ahave lso (by phage display technology) isolated sequences encoding allergen-specific IgE and characterized such proteins in order to define the diversity of recognition of allergens as it occurs in human allergy.
Figure 1. Evolution paths taken by genes encoding IgE with an origin in three different germline genes. The number of mutations differing the various members of these clonotypes derived from different germline genes are shown next to the arrows. (Andréasson et al., 2006)
|Figure 2. Two similar rearrangements identified in two different individuals (A3 and A4). These rearrangements, that due to their similarity shared a common specificity, originated from the same VH and JH genes but used different D-genes. Despite the differences in D gene usage, the rearrangement resulted in almost identical CDRH3 sequences. Several mutations were also shared or very similar (stars) in-between the different clonotypes as they evolve in vivo, further supporting the notion that they evolved their affinity for the same antigen. (Andréasson et al., 2006)|
In order to address the composition of allergen-specifi recognition of IgE, we cloned genes encoding heavy chain variable domains as found in the IgE with genes encoding human kappa and lambda light chain sequences. Combinatorial antibody libraries have been made and allergen-specific antibody fragments were selected by phage display technology.
1. The antibody repertoire targeting the major timothy allergen Phl p 5 has been characterized (Andréasson et al., 2006; Persson et al., 2007).
Figure 3. Evolution of a clonotype derived from the IGHV5-51 germline gene that has been shown to encode a known specficity, namely Phl p 5. This particular clonotype amounted to approximately 9% of all transcript in the IgE-encoding transcriptome found in peripheral blood B cells of an allergic individual. The numbers next to the arrows indicate the number of nucleotide substitutions that differ the various members in this proposed evolution pathway. (Andréasson et al., 2006)
2. To further assess the make-up of the IgE-encoding transcripome, we selected specific binders for additional timothy pollen allergens, namely Phl p 1, Phl p 2, Phl p 4, Phl p 6 and Phl p 11 (Persson et al., 2007).
Altogether, we have developed a unique panel of human IgE specificities targeting six different groups of grass pollen allergens, a probe resource for future assessment of the recognition of allergens by the human humoral immune system. Collectively they represent the first extensive set of an Anti-Allergome Resouce, i.e. sets of IgE representing the human antibody reponse that can interact with the much more extensively characterized Allergome, the set of allergenic proteins that induce type I hypersensitivity reactions by activating effector cells of the immune system that carry IgE.
We recently isolated human antibody fragments against the timothy allergen Phl p 2 (Persson et al., 2007) from an antibody library with an origin in the IgE-encoding transcriptome. Interestingly, the sequence of this scFv was very similar to a set of other antibody fragments specific for the same allergen but derived from a library developed from transcripts from another subject (Persson et al., 2008). Furthermore, some transcripts encoding IgE VH in other individuals allergic to grass pollen allergens were very similar in sequence, too. It thus appear that a common sequence motif is involved in the development of specific antibodies targeting a major epitope in the allergen Phl p 2. These findings extend the observation of existence of restricted antibody repertoires (previously defined in e.g. the response against haptens, viral and bacterial antigens, xenoantigens and autoantigens) also to human antibody repertoires targting allergens.
We have demonstrated that molecular library technology can be used to unravel molecular details of IgE as it occurs in allergic disease. It can also be used to develop unique research reagents targeting allergens. We envisage that such knowledge and reagents will help us understand allergic disease and to improve treatment, diagnosis and prevention of such diseases in the future.
Dr. Mats Ohlin
Dept. of Immunotechnology
Medicon Village (Building 406)
S-223 81 LUND
telephone: +46-46-222 4322
Page Manager: Mats Ohlin
Last updated: 2013-05-14
Phage display: a short introduction