F. Audibert

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.

Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muramyl peptide

Synthetic antigens employed in experimental synthetic vaccines are generally small haptenic peptides. Therefore, effective immunization with these antigens usually requires the use of an immunogenic carrier. Tetanus toxoid has been proposed for use as a carrier in future synthetic vaccines due to its high immunogenicity and acceptance for human use. Previous studies employing standard hapten/carrier systems such as DNP/KLH have demonstrated, however, that an epitope-specific suppression occurs when mice previously primed with carrier are subsequently immunized with an haptenic epitope conjugated to the same carrier. These same studies have shown that Bordetella pertussis vaccine administered at the time of carrier priming abrogates epitopic suppression. In the present investigation, epitopic suppression was studied in a synthetic vaccine model employing tetanus toxoid as a carrier. Results from these studies indicated that mice primed with tetanus toxoid 1 month before immunization with a peptide-tetanus toxoid conjugate exhibited enhanced secondary anti-tetanus toxin responses but decreased anti-peptide responses. Furthermore, injection of pertussis vaccine or purified B. pertussis toxin or endotoxin at the time of carrier priming could block the establishment of epitopic suppression. Administration of B. pertussis components enhanced antibody responses to both the carrier and the synthetic peptides as compared with responses of control animals. In addition, administration of an adjuvant-active nonpyrogenic derivative of muramyl dipeptide. Murabutide, with carrier priming reduced epitopic suppression of anti-peptide responses. B. pertussis toxin or endotoxin administered to mice previously suppressed by carrier priming with the first injection of carrier-peptide conjugate overcame epitopic suppression with resultant titers of anti-peptide antibody equal to or greater than nonsuppressed controls. These results suggest that the use of adjuvants with future synthetic vaccines may contribute the additional advantage of overcoming epitopic suppression, thus permitting the use of common, well-tolerated carrier systems such as tetanus toxoid in synthetic vaccine preparations.

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.

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.

Synthetic immunoregulating molecules: A potential bridge between cytostatic chemotherapy and immunotherapy of cancer

The concept of immunomodulation as an adjunct to cancer therapy is based on ample evidence that a variety of agents augment the immune reaction to unrelated antigens in experimental animals and increase their resistance to tumors. The ever-growing list of such agents includes viruses, bacteria, fungi, and their products (Ciba Foundation Symposium, 1973). Following the pioneering work of Math6 with BCG (Math~, 1971), investigators have been very active in this field in recent years (Chedid et al., 1973; Lamensans et al., 1975; Lamoureux et al., 1976; Laucius et al., 1974; Leclerc et al., 1976; Mastrangelo et al., 1976; Old et al., 1959; Weiss et al., 1961; Zbar et al., 1970). Most, if not all of these studies, have been done with viable or killed microorganisms (Hersch et al., 1976; Israel, 1976; Pouillart et al., 1976), cell walls (Chedid et al., 1973; Gray et al., 1975; Yamamura et al., 1976a; Zbar et al., 1972), or very complex fractions such as raethanol extract residue (MER) (Perloff et al., 1977; Yron et al., 1973) or interphase material (IPM) (Lamensans et al., 1975). Some organisms such as BCG and Corynebacterium parvum have been shown to be dramatically effective in certain experimental models, but investigators are well aware of the fact that they contain a great variety of heterogenous antigens, some of which might cross-react with host tissues (Borsos and Rapp, 1973; Bucana and Hanna, 1974; Vandenbark et al., 1975). They are also endowed with undesirable pharmacologic side effects (Werner et al., 1977). Consequently, current attempts at isolating chemically well-defined fractions from whole bacteria have been given a high priority, with the justification that they may lead to preparations that retain important therapeutic effects but with diminished or no toxicity.

Antibody responses elicited by a polyvalent vaccine containing synthetic diphtheric, streptococcal and hepatitis peptides coupled to the same carrier

Three synthetic peptides copying fragments of the diphtheria toxin, the M protein of the streptococcus type 24 and the hepatitis B virus surface antigen (HBs) have been conjugated together to the tetanus toxoid. This polyvalent vaccine has been administered to mice. High antibody titers were obtained against the three antigens. No cross-reactivity could be observed between them as demonstrated by the ability of each peptide to inhibit only the antibodies against the natural M protein and the synthetic M protein peptide indicated that the avidity of the antibodies raised against a monovalent streptococcal vaccine were identical to those raised following injection of the polyvalent vaccine. Antibodies raised against the polyvalent streptococcal vaccine were also protective as shown by opsonophagocytic assays.

Production of anti-sporozoite antibodies in absence of response to carrier by coupling an MDP derivative to a malaria peptide-tetanus toxoid conjugate.

A synthetic peptide (pep) representing a portion of the Plasmodium knowlesi circumsporozoite protein attached to a tetanus toxoid (TT) carrier, has been shown to be immunogenic when delivered in saline with derivatives of the synthetic adjuvant, muramyl dipeptide (MDP). The present study was designed to determine if the degree of substitution of pep and of MDP derivatives on the tetanus toxoid (TT) carrier, as well as the choice of MDP derivative used play a role in determining anti-pep and anti-TT antibody levels. One of the MDP derivatives used in the conjugates was epsilon-amino-caproic Murabutide, since Murabutide which is currently in clinical trials cannot be conjugated. The results show that low doses of this derivative coupled with pep on TT can be used to stimulate high levels of circulating anti-pep antibodies without augmenting the anti-carrier response. In addition, anti-pep antibodies elicited in response to one of the conjugates were biologically active since they produced shedding of the circumsporozoite coat of live parasites.

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.

Influence of CONH2 or COOH as C-terminus groups on the antigenic characters of immunogenic peptides.

The influence of the presence of a terminal COOH or CONH2 on the antigenic characters of synthetic immunogenic peptides has been studied on a streptococcal synthetic vaccine model. The obtained results show that when a peptide amide is used, the antibodies raised specifically against the amide group recognize neither free COOH nor the parent protein. The carboxamide group is thus unsuitable as was postulated for raising antibodies which recognize the peptide bond.