Claude Carelli

Immunological castration by a totally synthetic vaccine: modification of biological properties of LH-RH after conjugation to adjuvant-active muramyl peptide.

Recently, we demonstrated that immunological castration of male mice can be obtained by immunization with Luteinizing Hormone-Releasing Hormone (LH-RH) directly coupled to NAcMur-L-Ala-D-isoGln-L-Lys (MDP-Lys) without carrier and Freunds Complete Adjuvant (FCA) but in the presence of Polyvinyl-Pyrrolidone (PVP). In the present report, we have observed that: (a) immunization by the conjugate, LH-RH-MDP-Lys, was very effective even in absence of PVP, and this conjugate was more active than other conjugates containing MDP coupled to LH-RH fragments; (b) a strong secondary response could be observed by the administration of free LH-RH suggesting that the endogenous secretion of LH-RH might elicit a boosting effect; (c) administration of MDP-Lys coupled to LH-RH decreased the pyrogenicity of the glycopeptide; (d) such a conjugation also decreased the hormonal activity of the antigen although it enhanced its immunogenicity. These results show that a conjugate (2000 dalton) of a decapeptide hormone with a synthetic adjuvant glycopeptide can induce immunological castration in mice after administration in saline. The immunopharmacological properties of the conjugate and its conditions of efficacy suggest that such an approach could find clinical application.

Synthesis of conjugates containing N-acetylmuramyl-L-alanyl-D-isoglutaminyl (MDP). Their use as hapten-carrier systems.

Abstract Synthetic muramyl dipeptide (MDP) is the minimal structure which can substitute for mycobacteria in Freunds complete adjuvant. This glycopeptide was conjugated to several protein carriers via carbodiimide or phenylisothiocyanate intermediates. The resulting conjugates were used as antigens for the induction of antibodies bearing specificity for muramyl dipeptide hapten. In addition, the phenylisothiocyanate method of conjugation was adapted for the covalent linkage of hapten to formalin-fixed erythrocytes. It was shown that (a) MDP becomes immunogenic when linked to a carrier and that antibodies can recognize free synthetic MDP; (b) conjugation procedures do not abolish the adjuvant potential of the glycopeptide.

Analysis of the antigenic relationship of various derivatives of N-acetyl-muramyl-l-ala-d-isoglutamine (MDP), using anti-MDP antibodies

Antibodies to N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) were produced in rabbits by injection of MDP conjugated to various carriers [bovine gamma globulin (BGG), methylated BSA or sheep erythrocytes]. The anti-MDP was assayed by a direct enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase linked to MDP-Lys. Various derivatives of MDP were employed in an inhibition of ELISA for analysis of specificity of antibodies and study of the relationship of configuration to biological activity. The results confirmed previous findings that MDP alone is not immunogenic but can act as a hapten when conjugated to carriers. The antibodies were shown to be primarily directed against the muramyl residue. Modifications of this region of MDP yielded derivatives with weak reactivity against anti-MDP, while some changes of other regions had no effect on its antigenicity. Optical isomers of MDP had reduced activity as compared to MDP and polymeric MDP was a strong inhibitor. The structure and function relationship is discussed for some derivatives.

Decrease in pyrogenicity of muramyl dipeptide after coupling with luteinizing hormone-releasing hormone.

Muramyl dipeptide (MDP) and its adjuvant active derivative lysine‐MDP (Lys‐MDP) have been demonstrated to be pyrogenic and to induce endogenous pyrogen (EP) production in vivo and in vitro. It has recently been shown that immunologic castration can be achieved in mice by immunization with luteinizing hormone‐releasing hormone (LHRH) directly conjugated by carbodilmide to Lys‐MDP, termed LHRH‐Lys‐MDP (cdi), or with a linear monomeric MDP‐linked molecule obtained by total synthesis, termed LHRH‐Lys‐MDP (s). These preparations were tested in the rabbit for their capacity to induce fever and were found to be devoid of pyrogenicity at dosage levels of Lys‐MDP that induced fever. This decrease of pyrogenicity of Lys‐MDP after coupling to LHRH seems to be related to the structure of the conjugate because the derivative LHRH‐LysNH2‐MDP exhibited the same pyrogenic activity as the free glycopeptide. Surprisingly, nonpyrogenic LHRH‐Lys‐MDP induced production of EP and interleukin‐1 (IL‐1) in vitro and increased in vivo modifications of metal levels attributed to the action of IL‐1. Moreover, LHRH‐Lys‐MDP reduced the pyrogenic effect of an exogenous dose of EP.