Immunogenicity of dinitrocarboxyphenylated melittin: The influence of C‐terminal chain shortening, N‐terminal substitution and prolin insertion at positions 5 and 10

Abstract

Peptides derived from the bee-venom melittin were fitted with the haptenic group dinitrocarboxyphenyl (Dncp) and tested in out-bred guinea pigs for immunogenicity by measuring the IgG anti-Dncp antibody response by ELISA. Dncp-conjugates comprising virtually the entire melittin proved to be strong immunogens producing antibody responses comparable to those of proteins. Weak responses were obtained with considerably shortened sequences. Conjugates with N-terminal Dncp gave markedly reduced antibody responses compared to peptides with C-terminal Dncp. An N-terminal biotinyl substituent abolished the immune response whereas N-terminal lauryl and caprylyl had little effect. Insertion of L-proline into a hexadecapeptide conjugate abolishing the possibility of helix formation gave an immunogen to which individual animals clearly responded on a low level. Oligomerisation, but not the cytolytic activity of melittin peptides, may contribute to the immunogenicities observed.

Molecular parameters in melittin immunogenicity

“…A reference immunization was done with melittin (1± 26)K(Dncp)G, MD, previously synthesized and shown to give ELISA titres D r in guinea pigs of between 10 4 and 10 6 [13]. The data found for MD in the present experiment are shown together with those from the truncated peptide M(13±26)D in Figure. 1.…”

“…It is evident and originally unexpected that the truncated peptide still elicits a considerable antibody response. The expectation was that in M(13±26)D a marked decrease in immunogenicity should become noticeable because previous data have shown that chain shortening at the C-terminus down to M(1±19)D did not remove guinea-pig immunogenicity [13] and that therefore a centrally …”

“…Polystyrene microtitre plates (Dynatech) were coated with N 1 -Dncp±diaminohexane and used as described before [13,14]. The detecting antibodies goat anti-guinea pig IgG (H L) alkaline phosphatase and goat anti-mouse IgG (H L) alkaline phosphatase were obtained from Jackson Immunoresearch Laboratories, West Grove, PA. Titres were expressed as the highest reciprocal dilution (D r ) of the antisera giving an absorbance of 1.0 after 30 min incubation with 4-nitrophenylphosphate (1 mg/ml in 0.05 M carbonate buffer ± 1 mM MgCl 2 , pH 9.8).…”

“…GOHI-guinea pigs (out-bred, female, 250 g) from BRL, Ltd, Fu È llinsdorf, Switzerland, and Balb/c mice (female, 8±12-weeks-old) from IFFA, Credo, SaintGermain sur l'Arbresle, France, were immunized with Dncp±peptides as described before [13,10].…”

“…Other parameters of interest were the effect of large substituents present within T-cell epitopes on immunogenicity and the effect of chain shortening at the N-terminus. As recently published [13] immunogenicities of the peptides were studied by using melittin-derived peptides fitted with a single 2,4-dinitro-6-carboxylphenyl (Dncp) haptenic group at the C-terminal end. The haptenic structure serves as an effective antigenic determinant regularly selected by B-cells for antibody production.…”