Identification of adjuvants that enhance the therapeutic antibody response to host IgE.

In the development of a novel vaccine against atopic allergies, we have screened for adjuvants that enhance the therapeutic antibody response against self immunoglobulin E (IgE). The response against self IgE is induced by administration of a vaccine antigen, which contains both self and non-self IgE regions, together with an adjuvant. We evaluated five commonly used adjuvants; Freund's, aluminium hydroxide, ISCOMs, Montanide ISA 51 and Montanide ISA 720, and found that the mineral oil-based adjuvants; Montanide ISA 51 and Freund's induced at least 5-10-fold higher anti-self IgE titers than any of the other candidates. However, with one exception, Alum, the immune responses against the carrier, i.e. the non-self regions, were similar for all adjuvants, indicating that the ability to induce responses against self and non-self antigens differ among adjuvants. The responses against non-self IgE were more than 50-fold higher than antibody responses against self IgE in both the Freund's and Montanide 51-administered animals, indicating that the response against self molecules is markedly inhibited by tolerance-inducing mechanisms. Co-administration of Montanide ISA 51 with immuno-stimulatory substances from bacteria; muramyldipeptide (MDP), monophosphoryl-lipid A (MPL) or a formyl-methionine-containing tripeptide (fMLP), did not elevate the anti-self IgE response. Hence, adjuvants based on pure mineral oil without additional immuno-stimulatory substances appear to be the best adjuvant candidates in therapeutic vaccines aimed at regulating the in vivo levels of self-proteins.

Cloning of IgE from the echidna (Tachyglossus aculeatus) and a comparative analysis of epsilon chains from all three extant mammalian lineages.

In continuation of our evolutionary studies of immunoglobulin (Ig) expression, we present here the cloning of IgE from a monotreme, the short-beaked echidna (Tachyglossus aculeatus). Including echidna IgE, 15 epsilon chain sequences have been isolated and each of the three mammalian lineages (placentals, marsupials and monotremes) is now represented by at least two sequences. Phylogenetic analyses based on all available epsilon chains and a selection of other mammalian Ig isotypes (IgM, IgA and IgG) were generated using three different algorithms. The resulting trees strongly support the Theria hypothesis, which states that the monotreme lineage was the first of the three extant mammalian lineages to appear in evolution. Furthermore, to increase our understanding of IgE we have done a detailed comparative analysis, with focus on primary structure, potential N-glycosylation, charge distribution and conservation of residues in the putative receptor-binding site. The overall structure of IgE, i.e. four constant domains and the positions of putative disulfide-bridge formations, are conserved, as is an N-glycosylation site in the third constant domain. An increased homology was observed in the putative receptor-binding site, which suggests an important function for the IgE/Fc epsilon RI interaction. IgE has been found exclusively in mammals, but it is present in all extant mammalian lineages. This, together with the overall conservation of structure, indicates that IgE appeared as a separate isotype early in mammalian evolution and that structural maintenance may have a selective advantage.

Generation of therapeutic antibody responses against IgE through vaccination.

IgE is the central mediator in atopic allergies such as hay fever, eczema, and asthma; therefore, it is a prime target in the development of allergen-independent preventive treatments. We describe an active immunization strategy that has the potential to reduce IgE to a clinically significant extent. The active vaccine component is a chimeric IgE molecule, Cepsilon2-Cepsilon3-Cepsilon4. The receptor-binding target domain, Cepsilon3, is derived from the recipient species, whereas the flanking domains, Cepsilon2 and Cepsilon4, are derived from an evolutionarily distant mammal. The flanking domains have dual functions, acting both as structural support for the Cepsilon3 domain and to break T cell tolerance by providing foreign T cell epitopes. The efficacy of the vaccine was studied in an ovalbumin-sensitized rat model. Vaccination resulted in antibody responses against IgE in all rats and in a substantial reduction in serum IgE levels in three out of four strains. The skin reactivity upon allergen challenge was significantly reduced in vaccinated animals. The vaccine appears to be safe to use as an antigen. No cross-linking activity was observed in sera of vaccinated animals, and the response to vaccination was reversible with time. Our results suggest that active immunization against IgE has the potential to become a therapeutic method for humans.