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.

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In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in ∼36% of the platypus VH sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction.

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Based on the analysis of a panel of variable (V) region sequences from the Australian duck-billed platypus and the Australian short beaked echidna, the monotremes were found to express a highly diversified Vk repertoire. High variability was observed both in sequence and in length of all three CDR regions. However, all monotreme sequences were found to form a separate branch on a distance tree, and the monotremes appear to express only two Vk gene families. The appearance of all Vk gene segments in one branch on the distance tree gives further support for the notion that deletions of entire V region clans or families, followed by successive rounds of gene duplications may be a relatively common phenomenon during vertebrate evolution. Four different constant region sequences were also identified and a preferential use of certain J segments to each constant region was observed. A more detailed picture of the locus was obtained by analysis of genomic DNA by Southern blot and PCR. The organization of the lambda locus involves multiple V and several constant region genes with one or several joining segments positioned upstream of each constant region, similar to the organization in mouse and man. An mRNA frequency analysis shows that the k light chain accounts for more than 90% of the light chain transcripts in the spleen. The abundance and the high variability indicate that light chain diversity at the k locus contributes significantly to the antigen-binding repertoire in monotremes. A high k to j light chain ratio also indicates that variability in the CDR regions is more important for the repertoire size than the total number of V gene families.