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Tripalmitoyl-S-glycerylcysteinyl lipopeptides are B-cell and macrophage activating and may be used as low molecular weight immunogens of considerable potency and even as vaccines when conjugated with suitable epitopic structures. Selected lipopeptides carrying single Dnp haptens were found to evoke mild passive cutaneous anaphylaxis in guinea pigs sensitized against Dnp. The reactions were observed after intravenous injection whereas intradermally applied antigen was negative. The anaphylactogenicity seems unrelated to micelle or aggregate formation of the insoluble peptides which require lecithin additions as well as sonication to become solubilized. The dinitrophenylated lipopeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-lysine produced toxic reactions which were not observed with the lipopeptide devoid of Dnp. Dinitrophenylated tripalmitoyl-S-glycerylcysteiny-1,6-diaminohexane and tripalmitoyl-S-glyceryl-cysteinyl-lysine did not show these toxic reactions.

Two deca-L-lysine conjugates, one carrying ten, the other carrying six D-benzylpenicilloyl haptenic groups as epitope models, were used as elicitors of passive cutaneous anaphylaxis in guinea pigs. It was found that equal haptenic doses yielded virtually indistinguishable responses, i.e., the potencies were close to 10:6. Interpretation of these data appears possible by using established immunochemical concepts of preequilibria and equilibria in free solution as starting point. The discussion is simplified due to the fact that the two conjugates being decavalent and hexavalent, respectively, in free solution, are both hexavalent on cell membranes. It was found that the clusters of haptenic groups displayed by the conjugates enhance the affinity for antibody binding and that the haptenic densities are directly related to potencies. Epitope clusters of enhanced affinity interacting with cell-bound antibody may be significant and useful in a number of ways.

N1-DNCP-N6-lactobionoyl-1,6,hexanediamine, which is a potent anaphylactogen when i.v. injected into anti-DNCP sensitized guinea pigs, was shown to be also anaphylactogenic when administered intradermally into guinea pigs passively sensitized by i.v. injection of anti-DNCP antiserum. The compound, which is monohaptenic conjugate with the DNCP group as haptenic part and the carbohydrate moiety as auxiliary group, was found to be unable to precipitate high-titered anti-DNCP antisera. This precludes formation of simple, functionally oligovalent associates. Since many monohaptenic anaphylactogens with various hydrocarbon chains as auxiliary groups do not induce anaphylaxis when given intradermally, the anaphylaxis, demonstrated after intradermal antigen application, allows a biological distinction between the lactobionoyl and similar conjugates, and the hydrocarbon-bearing anaphylactogens.

Conjugates carrying a single 2-carboxy-4,6-dinitrophenyl hapten and as auxiliary group various thiazolidine substituents related to penicilloyl, were prepared and studied in guinea-pig passive cutaneous anaphylaxis (PCA). Included are monohaptenic model conjugates with discrete hydrocarbon auxiliary groups. One of the conjugates with the 3-formyl-2,2-dimethyl-1,3-thiazolidine-(4S)-oyl auxiliary group, characterized by the blocked chemical reactivity of the thiazolidine, was an anaphylactogen. This rules out formation of oligohaptenic antigens via chemical conjugation as a step in the PCA reactions observed. Rather, the non-covalent interactions of the auxiliary group, which has very strict structural requirements not yet mapped out in full, are necessary. Data from the model conjugates show that planar ring structures in conjunction with unsubstituted carboxyl groups constitute effective auxiliary groups. Benzylpenicilloyl antigens may act as monovalent elicitors of immediate reactions when the antibody population is predominantly directed against the benzyl side-chain. Under these conditions the benzyl residue is expected to serve as the haptenic part and the thiazolidine moiety as the auxiliary group.