Pierre Lefrancier

Distinctive adjuvanticity of synthetic analogs of mycobacterial water-soluble components

Abstract Synthetic N -acetyl-muramyl- l -alanyl- d -isoglutamine represents the minimal structure capable of duplicating the activity of mycobacteria in Freunds complete adjuvant. In contrast to mycobacterial adjuvant preparations, that function only in the form of water-in-oil emulsions, this compound and a second synthetic analog ( N -acetyl-muramyl- l -alanyl- d -isoglutamic acid) augment the humoral immune responses of mice equally as well as aqueous solutions. Whereas N -acetyl-muramyl- l -alanyl- d -isoglutamic acid administered to guinea pigs in water-in-oil emulsion has no effect, N -acetyl-muramyl- l -alanyl- d -isoglutamine induces delayed hypersensitivity to ovalbumin and azobenezene-arsonate N -acetyl- l -tyrosine and increases the level of antibody against ovalbumin. Under these conditions, challenge with the synthetic adjuvants themselves evokes no skin responses. Moreover, Freunds complete adjuvant sensitizes guinea pigs to tuberculin and to native mycobacterial water-soluble adjuvant but not to the synthetic analogs.

Correlation of structure and adjuvant activity of N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), its derivatives and analogues. Anti-adjuvant and competition properties of stereoisomers.

Abstract The synthetic N-acetyl muramyl-dipeptide (MDP) 1 has been shown to be fully adjuvant active in a water-in-oil emulsion; we now report a study on the adjuvant activity of 1O derivatives and analogues of MDP under similar conditions. NaBH 4 reduction of MDP 1 leads to the inactive muramicitol dipeptide 2 ; β-elimination gives the lactyl-dipeptide 3 , which seems to inhibit adjuvant activity. Shortening of the lactyl side chain of MDP gives nor-MDP, 4 , which is less active. Amongst the analogues in which one amino acid of the dipeptide moiety is replaced by another one, the L-Ser analogue 5 is fully active, whereas replacement of L-Ala by Gly or of D-iso-Gln by D-Glu-OH, or D-Glu (OMe) 2 , or D-Glu (OMe)-NH 2 gives less active compounds ( 6 , 8 , 9 , 1O ). The diastereoisomer 7 where L-Ala is replaced by D-Ala, shows an anti-adjuvant activity.

In vitro spleen cell responsiveness to various analogs of MDP (N-acetylmuramyl-l-alanyl-d-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice

Abstract N -Acetylmuramyl- l -alanyl- d -isoglutamine (MDP), a synthetic immunoadjuvant, was incubated with spleen cells of DBA/2 or Balb/c mice and optimal responses were obtained after 4 or 5 days of culture in a serum-free medium supplemented with 2-mercaptoethanol. In contrast, lymphocytes of (C57B1/6 × AKR)F 1 hybrids responded weakly under the same conditions. The results reported here show that like in the case of DBA/2 and Balb/c strains, spleen cells of Swiss mice and of inbred AKR and CBA mice could be stimulated in vitro whereas C57B1/6 and LPS-refractory C3H/He mice did not respond. Fourteen synthetic MDP analogs (eight known to be adjuvant active and six devoid of activity) were tested in DBA/2 high-responder mice. A good correlation was observed between in vitro stimulation and the presence or absence of adjuvant activity in vivo of these compounds.

A comparative 1H-N.M.R. study of MurNAc-l-Ala-d-iGln (MDP) and its analogue murabutide: Evidence for a structure involving two successive β-turns in MDP

The active principle, MurNAc-L-Ala-D-iGln (MDP), of complete Freunds adjuvant and its analogue, MurNAc-L-Ala-D-Gln-OnBu (murabutide), which express immunomodulatory as well as other biological properties, have been studied by 2D-1H-n.m.r. spectroscopy at 500 MHz. The results suggest the presence in MDP of two successive turns involving the MurNAc-L-Ala and L-Ala-D-iGln moieties, respectively, whereas only the former turn persists in murabutide. This turn mimics the type II beta-turn found in L-D depsipeptides, whereas the other is a typical type II beta-turn for L-D peptides.

Enhancement by muramyl peptides of the protective response of interferon-α/β against encephalomyocarditis virus infection

The use of interferon-α (IFN-α) in the treatment of infectious diseases has shown limited efficacy and dose-limiting toxicity. We have selected safe immunomodulators of the muramyl peptide family with the potential of enhancing the efficacy of IFN-α without resulting in increased toxicity. One of these synthetic muramyl dipeptide (MDP) derivatives, namely murabutide which is in a clinical stage of development, has been recently found to synergize with IFN-α2a in the selective induction of anti-inflammatory mediators and to enhance the biological activities of the therapeutic cytokine. The present study was performed to assess the antiviral activity of such muramyl peptides and a possible potentiation of the antiviral activity of IFN-α/β by associated therapy using the classical assay of Encephalomyocarditis virus (EMCV) infection. In vitro, pretreatment of Moloney Sarcoma virus (MSV)-transformed cell line with MDP derivatives followed by treatment with IFN-α/β showed a synergistic protection against the cytopathogenic effect of a subsequent EMCV infection. None of the MSV cultures could be protected by stimulation with muramyl peptides alone. In vivo, all of the muramyl peptide derivatives tested were found to be more potent than the parent molecule MDP in inducing protection against death or in the prolongation of the mean survival time of infected mice. Sequential administration of suboptimal doses of exogenous IFN-α/β and muramyl peptides established a strong antiviral state and considerably improved the protective effect of the cytokine, frequently leading to an abortive infection. Our findings suggest that combination therapy with safe muramyl peptides and IFN-α/β could constitute a highly effective and new regimen for the treatment of viral infections in humans.

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.

Dissociation of immunostimulant activities of muramyl dipeptide (MDP) by linking amino-acids or peptides to the glutaminyl residue

Abstract N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide or MDP) is the minimal structure required for substituting mycobacteria in Freunds complete adjuvant. It is an adjuvant when injected in saline, protects mice non-specifically against infection, and enhances thymidine incorporation of lymphocytes. In this report it is shown that the linking of L-Lys, L-Ala-D-Ala, L-Lys-D-Ala or L-Lys-L-Ala to MDP permits the dissociation of anti-infectious activity from adjuvant activity. The optical configuration of the added residues plays the major role in this dissociation. It can be noted that the muramyl tetrapeptide MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala mimicking the natural structure is devoid of anti-infectious activity.

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.