Molecular parameters in melittin immunogenicity.

Based on immunogenicity studies, two T-cell epitopes in melittin were found to be functional in guinea pigs, one being centrally located, the other one residing in the C-terminal chain. In Balb/c mice only the central epitope was found to be active. A human T-cell clone was found by T-cell proliferation studies to employ strictly the C-terminal chain. Truncation of melittin peptides at the N-terminus did not markedly affect the capacity of guinea pigs to develop anti-IgG responses towards peptidic epitopes and towards a C-terminally attached haptenic group. Attachment of various substituents inside and outside the T-cell epitopic areas had no marked effect on antibody responses. In contrast, the substituents positioned within a T-cell epitope abolished T-cell proliferation. This difference between whole animal data and cellular in vitro responses is presently not understood.

Immunogenicity studies with haptenated melittin peptides: implication for membrane involvement during the recognition step.

Melittin peptides carrying 2,4-dinitro-6-carboxyphenyl (Dncp) haptenic groups regularly evoked anti-hapten IgG responses in mice or guinea pigs when the hapten was C-terminally attached. Single haptens on the N-terminal helix in several positions gave poor or no responses in the early stages but adequate titres after prolonged immunization. Peptides with Dncp at the C-terminus as an invariant feature and a second Dncp in various positions along the peptide chain did not fail to produce adequate responses. The hampering effect is not due to a defect at the T-cell level but involves the recognition step on the B-cell. It is implied that the haptenic interaction with the paratope of the recognizing immunoglobulin on the B-cell involves the cell membrane in an important way. It is also suggested that late antibody responses should not be overlooked during the development of proteinaceous immunogens for vaccination.

Cross reactive anti-tetanus and anti-melittin Fab fragments by phage display after tetanus toxoid immunisation.

Amino acid sequence homology between tetanus toxoid, a common vaccine and melittin, a bee venom allergen provided us with two antigen models for studying the production of cross reactive antibodies after immunisation. Analysis of the serum of an atopic patient recently boosted with tetanus toxoid revealed the presence of anti-melittin and anti-tetanus antibodies. From this donor a phage display Fab library was constructed five days after vaccination. Screening of this library either with melittin alone or with tetanus toxoid followed by melittin allowed the isolation of Fab fragments specific to melittin but also Fab fragments which cross react with melittin and tetanus toxoid. Amino acid analysis revealed diversity in the heavy and the light Ig chains of the melittin specific and of the cross reactive clones. Interestingly we found that the light chain recognised melittin whereas the heavy chain preferentially bound to tetanus toxoid suggesting that cross reactivity may be due to the different binding specificities of the individual Ig chains.

Basic aspects related to penicillin-allergy skin testing: on the variability of the hapten-paratope interaction.

Ampicillin and benzylpenicillin conjugated to human serum albumin were used as immunogens in order to obtain antihaptenic IgG responses in outbred guinea pigs according to different schedules, all involving complete Freund's adjuvant. The individual responses were characterized by ELISA and by ELISA inhibition using ampicillin, benzylpenicillin, and carbenicillin peptidic conjugates for coating and for inhibition. In several instances, drastically reduced cross-reactivity and even its absence were observed, although the penicillin antigens differ only in the side-chain. The notion that the invariantly present thiazolidine ring will always provide significant binding to antibodies against all penicillins differing only in the side-chain has to be dropped. The experiments were performed in relation to newer findings of clinical penicillin-allergy skin testing which suggest that benzylpenicillin-based reagents alone are not able to detect or predict all reactions against semisynthetic penicillins. The experimental evidence here obtained corroborates this conclusion.