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Certain immunopharmacological activities of muramyl peptides have been associated with inflammatory and undesirable side-effects typically observed following the administration of the prototype molecule muramyl dipeptide. This activity is now demonstrated not to be linked to a direct activation of inflammatory processes in endothelial cells. Neither MDP nor other structural derivatives were able to induce inflammatory cytokines release or E-selectin gene expression in cultured human umbilical vein endothelial cells. However, oral administration of muramyl peptides has been reported to induce certain biological effects, including the downregulation of anamnestic, antigen-specific IgE responses, which are not observed following parenteral administration. We elaborate on these findings and extend them to show the efficacy of a new muramyl peptide in suppressing polyclonally induced serum IgE levels in anti-IgD-treated mice. The comparative effects of muramyl peptides, selected for clinical development, on the induction of cytokines in human whole blood are then presented at the level of mRNA accumulation and protein secretion. Moreover, the cytokine profile induced in vitro and in vivo by the combination of the safe immunostimulant, Murabutide, with interferon-alpha is examined. This combination reveals a selective and beneficial synergistic activity and induces anti-inflammatory cytokines in the absence of synergistic toxicity. The potential and the implications for the use of a therapeutic combination of an immunostimulant with a cytokine are discussed.

Activation of the cellular transcription factor nuclear factor-kappa B (NF-kappa B) by cytokines and other immunostimulants has been tightly linked with enhanced replication of human immunodeficiency virus-type 1 (HIV-1) in infected cells. Various immunomodulators are currently being examined in animal and human trials for their suitability as adjuvants in potential vaccines against acquired immunodeficiency syndrome (AIDS). It may prove to be beneficial to select adjuvants that do not induce NF-kappa B activation and particularly if the vaccines are to be aimed at seropositive individuals. We have examined a battery of synthetic immunostimulants of the muramyl peptide family for their ability to activate NF-kappa B in human and mouse cell lines. In this report, we demonstrate selective activation of NF-kappa B in different cell lines and by different muramyl peptides possessing immunostimulatory activities. The mechanism of such activation is apparently via production of reactive oxygen intermediates (ROI) since pretreatment of cells with antioxidants blocked subsequent activation of NF-kappa B. However, among all the molecules tested only one lipophilic, non-pyrogenic adjuvant active muramyl peptide showed a complete lack of NF-kappa B activation in all cell lines tested. This molecule could well become the adjuvant of choice in future AIDS vaccines.

N-Acetylmuramyl-L-alanyl-D-isoglutamine and four other synthetic adjuvants that are structural analogs of part of the mycobacterial peptidoglycan monomer are shown to enhance the nonspecific immunity of mice infected by Klebsiella pneumoniae. These compounds are active by various routes, including oral administration; they are also effective when administered after challenge. Of the seventeen other analogs tested, none is able to increase significantly resistance to infection, although seven of these molecules are adjuvant-active in saline. Previous results have shown that in contrast to lipopolysaccharides, these synthetic adjuvants are devoid of immunogenicity, mitogenicity, and toxicity in normal or adrenalectomized mice.

Natural and synthetic adjuvants of microbial origin were compared for their capacity to potentiate the induction of experimental autoimmune thyroiditis (EAT) with the autoantigen mouse thyroglobulin (MTg). Regardless of the immunomodulator used, severe thyroiditis was observed only in EAT-susceptible strains of the k haplotype and not in EAT-resistant strains of the d haplotype. Compared to phenol-extracted lipopolysaccharide, a potent adjuvant for enhancing EAT induction, phthalyl-substituted, detoxified lipopolysaccharide, even at doses 15- to 50-fold greater, led to only low anti-mouse thyroglobulin titers and mild thyroid infiltration. The synthetic adjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and three of its analogs, N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanyl-D-glycerol mycolate (MDP-L-Ala-Glyc-Myc), N-acetylmuramyl-L-alanyl-D-glutamyl-(decyl)methyl ester [MDP(decyl)methyl], and N-acetylmuramyl-L-alanyl-D-glutamine-alpha n-butyl ester [MDP-(Gln)-OnBu], designated murabutide, were tested in incomplete Freund adjuvant or in saline. In incomplete Freund adjuvant, MDP-L-Ala-Glyc-Myc was inefficient in inducing EAT, murabutide induced very mild involvement, and MDP and, more so, MDP(decyl)methyl were active but to a lesser degree than CFA. When saline was used, low levels of thyroid infiltration were observed in a few of the MDP-treated animals in only one experiment, whereas no lesions were observed when murabutide was used.

Two purified peptides extracted from E. coli or B. megaterium strongly stimulated the spleen lymphocytes of rabbits and of normal or nude mice. Both preparations can substitute for Mycobacteria in Freund's complete adjuvant. The peptidoglycan extracted from M. lysodeikticus by a similar procedure which lacks adjuvant activity, did not induce blast transformation. However, the monomere of the E. coli peptidoglycan was devoid of mitogenicity although it has also a marked adjuvant activity.

The adjuvant activity of two chemically well-defined bacterial products is reviewed: (1) lipopolysaccharides of gram-negative bacillis and their acylated detoxified derivatives, and (2) mycobacterial water-soluble fractions and synthetic analogues. Water-soluble adjuvant can substitute for mycobacteria in Freund's adjuvant, but if it is administered in saline, it has little activity. In contrast, lipopolysaccharide under the same conditions markedly increases the humoral antibody response. However, the use of lipopolysaccharide is limited by its toxicity. Water-soluble adjuvant treated with phthalic or succinic anhydride was shown to be an adjuvant when administered in saline. Furthermore, synthetic M-acetyl-muramyl-L-alanyl-D-isoglutamine also increased the humoral immune response when given in aqueous medium instead of in the usual water-in-oil emulsion. This compound, which has a small molecular weight, is not mitogenic, immunogenic, or toxic in mice, and was shown to have adjuvant activity even when given by the oral route.

Synthetic N-acetyl-muramyl-L-alanyl-D-isoglutamine, hereafter referred to as muramyl dipeptide or MDP, represents the minimal structure which can replace mycobacterial cells in Freund's complete adjuvant. This synthetic adjuvant can also enhance the humoral antibody response when administered to mice with an antigen in water by various routes including the oral route. The present paper reports some new results concerning the relationship between the structure and the adjuvant activity of MDP. Synthetic analogues of MDP were tested either in a water-in-oil emulsion in guinea-pigs or in saline in mice. The chemical modifications concerned essentially the D-glutamyl residue. The results obtained confirm the importance of maintaining the intact structure of the D-Glu residue and show that adjuvant activity can be abolished by certain modifications at the level of both carboxyl functions.

Muramyl dipeptide (MDP) is a small molecular weight synthetic glycopeptide (less than 500), which has been shown to be an immunoadjuvant, and to induce a biphasic febrile response in the rabbit--probably via the release of endogenous pyrogen--accompanied by a marked leukopenia. Macromolecularization by coupling to a synthetic carrier (MW approximately or equal to 60,000) potentiates the immunostimulant properties of MDP but also its pyrogenicity. The present study demonstrates that such a conjugate induced the release of endogenous pyrogen in vivo and in vitro at lower dosage levels than free MDP. Further experiments showed that there existed several differences between free and conjugated MDP. Thus, after intravenous administration of the conjugate, the fever pattern was monophasic with a prompt defervescence and not accompanied by leukopenia at dosage levels inducing similar increase in body temperature. In addition, when fever was recorded after intracerebroventricular administration, the increase in sensitivity was much greater in the case of free MDP than of MDP-A--L.

Specific anti-dinitrophenyl (DNP) response to DNP-conjugated L-glutamine60-L-alanine30-L-tyrosine10 (DNP-GAT) was obtained in GAT-responder mice by using synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as adjuvant. Significant levels of anti-DNP antibodies were observed during a secondary response to DNP-GAT, when both antigen and MDP were used for priming. In this system, MDP was able to prime the carrier-specific T cells but not the hapten specific B cells. The study of the isotypic pattern of the anti-DNP response shows that MDP stimulates only the appearance of specific anti-DNP IgG1 plaque-forming cells. Anti-DNP plaque-forming cells were stimulated in animals primed with DNP-GAT in Freund's complete adjuvant or in Maalox-pertussis and used as control IgG1, IgG2a, and IgG2b.

Modulation of myelopoiesis by three synthetic muramyl peptides was investigated in vivo. Two adjuvant-active compounds (N-acetylmuramyl dipeptide [MDP] and MDP-butyl-ester) elicited significant responses in DBA/2 mice characterized by a rise in the level of monocyte-macrophage colony-stimulating activity in serum, a proliferation of multipotential stem cells in the bone marrow, and an expansion of granulocyte-macrophage progenitors in the spleen. In contrast, the adjuvant-inactive stereoisomer MDP(D-D) induced only low levels of circulating colony-stimulating activity. Thus, MDP or MDP-butyl-ester injection could induce a greater number of macrophages and therefore enhance both specific and nonspecific immunity.

Successful human vaccination by synthetic malarial sporozoite peptides may depend on the choice of an appropriate carrier. Tetanus toxoid (TT) has been proposed because of its safe and widespread use in humans. Paradoxically, however, prior exposure to this toxoid vaccine could produce specific epitopic suppression against synthetic malarial peptides conjugated to this same protein as carrier. Indeed, we have previously reported that such a phenomenon can occur in the case of a synthetic vaccine made with a streptococcal peptide conjugated to TT. Our present study shows that similar results can be observed in mice preimmunized with TT 1 month before the administration of a conjugate containing TT and a Plasmodium knowlesi peptide. Analysis of the isotypic pattern of the antipeptide response showed that the immunoglobulin G1 (IgG1) subclass and especially the IgG2a and IgG2b subclasses were suppressed. In contrast, when a sporozoite peptide from Plasmodium falciparum was coupled to TT, the total antipeptide antibodies and particularly the IgG1 subclass were enhanced by preimmunization by TT. This increase of antipeptide antibodies was correlated with a greater ability of the sera to neutralize sporozoite infectivity. These results indicate that prior exposure to TT does not systematically impair the antibody response against a peptide administered as a peptide-TT conjugate.

The effect of a water soluble adjuvant (WSA) extracted from mycobacteria on the immune response to synthetic polypeptides has been studied in rabbits and inbred mice. WSA increased the antibody response of rabbits to the synthetic polypeptide poly(Tyr,Glu)-poly(DLAla)--poly(Lys) designated (T,G)-A--L when injected together with either incomplete or complete Freund's adjuvants (FIA or FCA). As compared to the respective controls, the effect of WSA was more pronounced when injected with FIA. No detectable antibody titre was observed in rabbits immunized with (T,G)-A--L and WSA administered in an aqueous medium. WSA had no enhancing adjuvant effect on the antibody response of C3H.SW high responder mice to (T,G)-A--L when given either with FIA or with FCA. However, the antibody titres of C3H/HeJ and SJL low responder mice to (T,G)-A--L were increased by WSA when injected in combination with FIA or FCA. The immune responses of mice to two other synthetic polypeptides poly(Phe,Glu)-poly(DLAla)--poly(Lys), designated (Phe,G)-A--L, and poly-(Tyr,Glu)-poly(Pro)--poly(Lys), designated (T,G)-Pro--L, were not affected by the addition of WSA. Thus, WSA "corrects" selectively the genetic defect of low responder mice to synthetic antigens.

The data reported here demonstrate that a preparation extracted from nonpathogenic mycobacteria such as Mycobacterium smegmatis and hereafter referred to as interphase material protected mice against Ehrlich ascitic carcinoma, L-1210 leukemia, and another syngeneic lymphoid leukemia. Furthermore, mice treated by this preparation were much less susceptible to endotoxins than when stimulated by BCG (bacillus Calmette-Guerin) or M. smegmatis cells. Moreover, guinea pigs treated by interphase material administered in Freund's incomplete adjuvant showed an increased immune response, yet their sensitivity to tuberculin was much weaker than that of controls sensitized with Freund's complete adjuvant. Finally, resistance to Columbia SK virus infection could be demonstrated when interphase material was administered to mice prior to virus challenge.

Delipidated lysozyme digests of Nocardia opaca, N. corallina, and N. rubra have been fractionated by Sephadex filtration. The mitogenic and adjuvant activities of the fractions thus obtained have been investigated. All fractions are mitogenic except the last fraction of N.rubra, but the N. opaca products induce a stronger stimulation of mouse spleen lymphocytes than the corresponding fractions of the two other species. The activity of the first Sephadex fractions of each strain has been compared to other mitogens (concanavalin A, lipopolysaccharide). All fractions are adjuvant, although one of them, the last Sephadex fraction of N. rubra, does not contain peptidoglycan; its activity must thus be attributed to another kind of molecule. Fractionation of the first Sephadex fraction of N. opaca by centrifugation in glacial acetic acid led to a separation of adjuvant and mitogenic activities.

Administered to guinea-pig, a synthetic compound, N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), has been previously shown to substitute for Mycobacteria in Freund's complete adjuvant. Moreover, MDP increases the humoral immune response even when administered in an aqueous solution to mice. In the present report, it was demonstrated that administered to guinea-pig in a water-in-oil emulsion, MDP or active analogues favoured the production of IgG2 antibodies against ovalbumin. In contrast, derivatives of MDP which had no adjuvant activity failed to induce this particular class of immunoglobulins. MDP without antigen, in contrast with LPS or FCA, did not induce changes in immunoglobulin levels in mice. Administered in mice with an antigen, MDP induces an increase of IgG1 although the immunoglobulin levels are lower than those observed after immunization with adjuvants injected in a water-in-oil emulsion.

The adjuvant effects of mycobacteria can be replaced by more chemically defined isolates of the cell walls including a water soluble fraction (WSA) and by the synthetic analog N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal structure required for adjuvanticity. These compounds can directly activate macrophages as determined by an increase in spreading and adherence and by an elevated synthesis of the enzyme collagenase. Moreover, this increase in collagenase production is modulated by enhanced production of prostaglandins that influences intracellular levels of cyclic AMP. In addition, both MDP and WSA induced macrophages to produce a biologically active mediator that triggers quiescent fibroblasts into active proliferation. It thus appears that a mechanism for mycobacterial adjuvant action as determined with MDP and WSA is via activation of macrophages, which may then precipitate a multiplicity of other reactions resulting in enhanced immune phenomena. Furthermore, the granulomatous and fibrotic reactions associated with mycobacterial infection may be a consequence of this direct activation of macrophages.

The influence of Murabutide, a muramyl peptide analog, on the response of mice to two synthetic hepatitis B virus antigens was compared with the activity of Al (OH)3 and of FCA. The synthetic peptides represented fragments either of the major component of the hepatitis B surface antigen (HBsAg) or of the pre-S region. They were used either conjugated to toxoid carrier or as a totally synthetic preparation, i.e. copolymerized with a streptococcal peptide. Our results demonstrated that treatment with Murabutide increased the levels of antibodies, modulated their specificity and allowed a better recognition of the natural antigens.

A decreased fibrinolytic activity induced by bacterial products and some muramyl peptides has been previously demonstrated in macrophages preparations. Since vascular endothelial cells are important for the fibrinolytic balance, we have studied the effects of MDP derivatives on cultured endothelial cells. The supernatant of MDP and murabutide treated cell cultures exhibited an increased fibrinolytic inhibitory activity when tested with urokinase. The MDP(D-D)-treatment had no effect. This increased inhibitory activity was detectable in the supernatant after a 6 h treatment and was suppressed by the addition of puromycin to the cell cultures. Furthermore, the endothelial cell culture supernatant also reduced the lytic activity of the human plasma plasminogen activator induced by venostasis. This was enhanced by MDP treatment of the cultures. These in vitro results suggested that adjuvant-active muramyl peptides may regulate the fibrinolytic balance at the vessel wall level. This could be of possible significance in the transendothelial cell migration where the role of plasminogen activator(s) has been involved.

The activity of Nocardia water-soluble mitogen (NWSM) and LPS were compared in several experimental systems, since both compounds are B-cell mitogens and polyclonal activators in vitro. The results reported here demonstrated that NWSM like LPS also has a strong adjuvant activity in vivo if administered in saline with a strong antigen (heterologous red blood cells) or even with a weak immunogen such as theta alloantigen. However, in contrast to LPS, NWSM administered to mice failed to induce in vivo proliferation of lymphocytes, polyclonal activation and PFC against syngeneic bromelain-treated erythrocytes and thymocytes. It is possible therefore, that different mechanisms may be responsible for adjuvant activity of NWSM and LPS.

The adjuvant effect of a synthetic peptidoglycan, muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine, MDP), was studied by using the anti-Tnp PFC and hemagglutinin responses of BALB/c mice to hapten-carrier conjugates. Administration of Tnp-OVA and MDP in saline to mice, followed 2 weeks later by a boost of Tnp-OVA in saline, led to significantly higher IgM and IgG anti-Tnp PFC and total anti-Tnp-hemagglutinin responses than those obtained in mice not treated with MDP in the initial immunization. A similar adjuvant effect by MDP on anti-hapten PFC responses was seen if mice were primed with KLH together with MDP and challenged with Tnp-KLH 2 weeks later. This apparent effect on carrier priming for helper function was confirmed and quantitated by double adoptive transfer experiments with graded numbers of spleen cells from KLH +/- MDP-primed mice and a fixed number of hapten-primed spleen cells from syngeneic Tnp-OVA immunized animals. These data suggest that at least one mode of action of the synthetic adjuvant MDP is via the enhanced stimulation of the helper T cell function.

Use was made of water-soluble adjuvant fractions with homologous sperm, sperm extract or sperm autoantigens to induce autoimmune aspermatogenic orchiepididymitis (AIAO) in guinea-pigs. Guinea-pigs were injected with soluble sperm extract or sperm purified autoantigen S emulsified in incomplete Freund adjvant (IFA) together with two synthetic watersoluble adjuvants, Mur-Nac-L-Ala-D-isoGlu et Mur-Nac-L-Ala-acide D-isoGlu. These animals developed lesions of AIAO, delayed hypersensitivity reactions and specific anti-spermatozoa antibodies. These lesions and reactions were comparable to the ones induced in a control group treated with the same spermatozoa extract or autoantigen S in complet Freund adjuvants. No lesion was observed in the absence of IFA, Other guinea-pigs received a mixture of spermatoza or purified sperm autoantigens, S, P, T. and of polyadenylicpolyuridylic complex either in saline or emulsified in IFA. The recipients of sperm or autoantigen T mixed with Poly A:U, with ou without IFA, displayed lsdions of AIAO; autoantigen S and Poly A:U in IFA, provoked AIAO but did not do it in the absenceof IFA.

The binding of the synthetic immunoadjuvant N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP, for muramyl dipeptide) to human and rabbit sera containing peptidoglycan (PG) antibodies was investigated. Studies were performed by employing the corresponding 14C or 125I-labeled compounds in Farr-type binding and inhibition assays. Whereas MDP did not react with naturally occurring or experimentally induced PG-antibodies, the analog of MDP MDP-L-Lys-D-Ala did bind to hyperimmune rabbit anti-PG sera, but not to the human sera tested. These studies indicate that the radioimmunoassays employed basically are applicable for the selection of nonimmumogenic MDP analogs possessing immunoadjuvant activity.

It has repeatedly been observed that various bacterial preparations could increase the host's resistance to tumors. It has also been shown that after nonspecific activation by BCG (bacillus Calmette-Guérin), peritoneal macrophages could inhibit in vitro the growth of neoplastic target cells. In the present study a fraction extracted from Myobacterium smegmatis and referred to as interphase material was tested in view of measuring its ability to activate macrophages in vitro and in vivo. This preparation was previously shown to protect mice against a syngeneic leukemia and to increase the immune response of the guinea pig. Other water-soluble adjuvants devoid of demonstrable antitumor activity in vivo were also assayed. The results argue in favor of a correlation between adjuvant activity and the capacity of activating macrophages. Moreover, interphase material administered in vivo consistently induced stronger and more persistent stimulations of macrophages than the other preparations assayed.

Murabutide (N-acetyl-muramyl-L-alanyl-D-glutamine-alpha-butylester), an MDP analogue, is a potential adjuvant for Human immunization. High levels of specific antibodies were obtained in Mouse and Guinea-Pig following administration with murabutide of low dosages of anti-hepatitis B viral vaccine containing the surface antigen (HBs). The effect of murabutide was enhanced without increasing the level of specific IgE by association with suboptimal dosages of aluminium hydroxide.

Adjuvants are largely used in human and veterinary medicine with conventional vaccines. Vaccines of the future could be synthetic immunogens which would require the addition of more immunomodulators. Muramyl peptides (MDP) are synthetic glycopeptides which can substitute mycobacteria in FCA and are also adjuvant-active in saline. A derivative, Murabutide, is currently undergoing clinical trials and has been found efficient in several experimental models of conventional and synthetic vaccines. For example, when a Plasmodium knowlesi sporozoite peptide conjugated to tetanus toxoid was administered in saline with Murabutide higher titers of biologically active antibodies were obtained than when the same conjugate was given in FCA. Coupling of MDP or derivatives to the immunogens enhanced their activity and allowed the use of carriers to be diminished or even avoided. In the same malarial model the linkage of a Murabutide derivative to the conjugate stimulated the response to the peptide and decreased the response to the carrier. Promising results were obtained when several peptides copying structures of different pathogens were associated in the same molecule by covalent linkage and administered with Murabutide in saline: biologically active antibodies were demonstrated to three of the corresponding pathogens.

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 carbodiimide 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.

The in vivo induction of colony-stimulating activity (CSA) by N-acetylmuramyl-L-alanine-D-isoglutamine has been demonstrated recently. In this study we increased our understanding of this property by testing muramyl peptides of several structures and activities for their capacity to induce CSA in vivo. A comparison of the anti-infectious and adjuvant activities of these molecules revealed no correlation between the capacities of these compounds to be adjuvant active and to induce CSA: all adjuvant-inactive compounds induced CSA, and certain adjuvant-active molecules did not induce CSA. In contrast, all anti-infectious compounds induced CSA, but the reverse was not true; some compounds devoid of anti-infection activity were able to induce CSA only if they were adjuvant active.

A wide range of adjuvants including alhydrogel, saponin, Corynebacterium parvum, DDAB, Pfizer CP-20,961, oil adjuvants and several MDP analogues have been compared for their adjuvant activity in protecting mice against lethal Trypanosoma cruzi infection following immunization with a T. cruzi 90K cell surface glycoprotein. Only saponin was found to be effective. Promotion did not correlate with the ability to promote a particular Ig isotype; however, saponin was unique in its ability to promote cell-mediate immunity against the 90K glycoprotein.

Muramyl dipeptide or MDP (AcMur-L-Ala-D-iGln) is a synthetic immunoadjuvant which can also enhance non-specific resistance to bacterial infections in mice, even by the oral route. By the use of several derivatives, it has been shown that neither adjuvanticity nor pyrogenicity was a perequisite for eliciting an increased resistance, and that unwanted pharmacological effects can be eliminated by minor chemical modifications. Moreover, some lipophilic analogs or derivatives obtained by linking the glycopeptide to a carrier were found to be more active than MDP. Their effectiveness also depended on the dose and the timing of administration, and varied according to the bacterial challenge. The most appropriately timed administration of MDP and derivatives was established between one and four days before the challenge. In some cases, MDP was protective even when injected one hour after the challenge, whereas with other immunostimulants such as lipopolysaccharides or BCG, a negative phase of higher susceptibility may occur under these conditions. MDP still enhanced resistance to bacterial infections in animals with a poor immune status, like newborns or adult mice under immunosuppressive treatment. Moreover, the protective activity was not impaired after repeated injections of large doses of MDP or other adjuvant analogs, a treatment which is known to inhibit specific immune responses.

The mechanism of adjuvant activity of the synthetic glycopeptide N-acetylmuramul-L-alanyl-D-isoglutamine or muramyl dipeptide (MDP) was studied using in vitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC). Addition of MDP to DBA/2 mouse spleen cell cultures resulted regularly in a 2 to 3-fold increase of PFC numbers/10(6) recovered cells (p less than 0.01). Supernates (SPN) from MDP-stimulated cultures added to standard spleen cell + SRBC cultures brought about even more important increases of PFC numbers (p less than 0.01 to p less than 0.001). SPN from cultures supplemented with MDP alone (without SRBC) were more active than those of cell + MDP + SRBC cultures, and SPN removed on day 3 of culture were more active than those of day 5. This activity of SPN was maintained accross an H-2 histocompatibility barrier. Although pretreatment of spleen cells with anti-theta antigen serum entirely suppressed the anti-SRBC PFC response in spite of the presence of MDP, SPN from these cultures were as active as SPN from normal spleen cell MDP-stimulated cultures. In contrast, pretreatment of spleen cells with specific rabbit anti-mouse macrophage serum entirely suppressed both anti-SRBC response and SPN activity. It was concluded that the target cell for MDP is the macrophage which releases factors ultimately acting on B cells through T cell mediation.

In a previous study we demonstrated that antiviral response against the coliphage MS-2 can be elicited by immunization with a synthetic antigen consisting of a conjugate (P2-A -- L) of a synthetic fragment (P2) of the virus coat protein attached to a synthetic polymeric carrier. The antiviral response was induced when the antigen was administered in complete Freund's adjuvant or when it was administered in incomplete adjuvant, provided that a peptidoglycan was covalently attached to it. In the present study we demonstrate the adjuvant effect of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) in this system. Immunization with a mixture of MDP and P2-A -- L brought about only slight enhancement in the titer of neutralizing antibodies, as compared to the immunization with P2-A -- L in saline. The best results were achieved when the MDP was chemically conjugated to P2-A -- L. This completely synthetic material, when administered in aqueous solution, yielded highly inactivating antiserum with a titer similar to that obtained with complete Freund's adjuvant in the absence of MDP. MDP-P2-A -- L elicited also a humoral immune response to MDP, but with much lower titer than that induced by complete Freund's adjuvant containing P2-A -- L only. It was also observed that the capacity of MDP-P2-A -- L to increase resistance against infection was more than a 100-fold greater than that of unconjugated MDP.

The ability of liposomal immunomodulators to restore abnormal macrophage tumoricidal activity has been studied. Macrophages from A/J mice have impaired responses in vitro to macrophage activating factor, gamma-interferon, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) or lipopolysaccharide when compared with macrophages from normoresponsive C57BL/6J mice. Liposomes containing a lipophilic muramyl dipeptide MDP-glyceroyl dipalmitate, macrophage activating factor or gamma-interferon restored tumoricidal activity to levels similar to C57BL/6J macrophages. Pretreatment of A/J mice with Corynebacterium parvum resulted in low levels of macrophage tumoricidal activity: treatment of C. parvum-induced A/J macrophages in vivo or in vitro with liposomal MDP-glyceroyl dipalmitate or liposomal macrophage activating factor resulted in normal levels of cytotoxicity. Macrophages from A/J mice were unable to phagocytose liposomes in vitro as rapidly or to the same extent as macrophages from C57BL/6J mice. The levels of cytotoxicity observed indicate that this is not a limiting factor for the induction of tumoricidal activity by liposomal immunomodulators.

MDP and some other synthetic glycopeptides which are endowed with adjuvant properties are also able to increase non-specific resistance of mice to bacterial infections. They are effective by various routes including the oral route, and this protective activity can be demonstrated in animals having a poor immune status.

N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP), is a synthetic immunoadjuvant analogue of a bacterial peptidoglycan subunit that has a definite pyrogenic effect in the rabbit. Some adjuvant-active derivatives such as murabutide [MDP(Gln)-OnBu] or murametide [MDP(Gln)-OMe] are not pyrogenic. Murabutide did not stimulate human or rabbit cells to release endogenous pyrogen (EP), but murametide induced EP production at the same dosage levels as MDP. Moreover, plasma from rabbits treated with murametide transferred into untreated recipients elicited a febrile response typical of EP fever and comparable with that induced by plasma from MDP-treated animals. Murametide not only inhibited the central effect of EP that is generated but also the effect of an extra dose of EP administered later by the intravenous route. Moreover, pretreatment of rabbits with murametide decreased fever responses induced by certain high-molecular-weight exogenous pyrogens as mediated through the production of EP.

Though highly efficient, Freund's adjuvant has serious limitations because it contains mineral oil and isomannide monooleate emulsifier (arlacel A). The present study describes the conditions under which a stable water in oil emulsion can be produced by using metabolizable peanut oil with arlacel. When mycobacteria are added, a potent emulsified oil adjuvant is obtained which increases the immune response to BSA and to influenza vaccine.