Adjuvant polyarthritis. V. Induction by N-acetylmuramyl-L-alanyl-D-isoglutamine, the smallest peptide subunit of bacterial peptidoglycan.

N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), an apparently nonimmunogenic bacterial peptidoglycan-derived small peptide, was found to induce a polyarthritis the rat similar to that induced by Freund's complete adjuvant when injected in the form of an oil emulsion. An oil emulsion of its isomer, N-acetylmuramyl-L-alanyl-L-isoglutamine, which unlike MDP has no immunostimulatory activity, failed to induce the disease.

Central pyrogenic activity of muramyl dipeptide.

Fever can be elicited in the rabbit by the intravenous administration of relatively large doses of a synthetic immunoadjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP). This response could be mediated by endogenous pyrogen because MDP has been shown to induce their production both in vivo and in vitro. The results reported here show that intracisternal injection of minute amounts of MDP could elevate fever without activating the release of endogenous pyrogen in the plasma or in the cerebrospinal fluid. Moreover, indomethacin inhibited hyperthermia produced by intracerebroventricular administration of MDP. Therefore, our findings argue in favor of a direct effect of the glycopeptide on the thermoregulatory centers besides its indirect effect through the production of leukocytic pyrogen. This molecule apparently represents the minimal requirement for the pyrogenicity of bacterial peptidoglycan because administration, even by the intracerebral route, of a mixture of muramic acid and of its dipeptide moiety did not elicit fever.

Opsonic antibodies evoked by hybrid peptide copies of types 5 and 24 streptococcal M proteins synthesized in tandem.

The protective immunogenicity of a hybrid peptide containing tandem copies of types 5 and 24 epitopes was investigated. Carboxy-terminal peptides of the cyanogen bromide-derived fragment 7 (CB7) of type 24 M protein were chemically synthesized, and then extended to include the first 20 residues of the amino-terminus of type 5 M protein. When emulsified in CFA and injected into rabbits without conjugation to a carrier, each of the synthetic hybrid peptides, designated S-M5(1-20)-S-CB7(23-35)C and S-M5(1-20)-S-CB(19-34), evoked opsonic antibodies against both types 5 and 24 streptococci without raising heart tissue-crossreactive immunity. These results suggest that tandem hybrid peptides may provide a new approach to the development of multivalent vaccines, not only to different serotypes of group A streptococci but perhaps also to a variety of other infectious agents.

Muramyl peptides. Variation of somnogenic activity with structure.

Sleep-promoting activities of muramyl dipeptide (MDP) (NAc-Mur-L-ala-D-isogln) and the naturally occurring muramyl peptide(s), factor S, have recently been demonstrated. We now have amplified our understanding of structural requirements for somnogenic activity. The effects of several analogs of MDP on rabbit slow-wave sleep are presented and these results are compared to the dose-response relationship for MDP. Some tentative conclusions as to structural requirements for somnogenic activity are presented; most notably, amidation of the free gamma-carboxyl of MDP and several of its analogs resulted in the loss of somnogenic activity. MDP also can induce febrile and immunostimulatory responses. In the present paper, we show that some analogs possess immunostimulatory and pyrogenic activity but not somnogenic activity, thus suggesting that these biological activities of muramyl peptides may, in part, be mediated by separate mechanisms.

Induction of colony-stimulating activity in mice by injection of liposomes containing lipophilic muramyl peptide derivatives.

Colony-stimulating factor (CSF) activity induction by lipophilic derivatives of three muramyl peptides, glyceryl dipalmitate-MDP derivatives, was studied in vivo and in vitro and compared to the activity of the same compounds incorporated within freeze-dried liposomes. Two lipophilic derivatives (MDP-GDP and MDPGBe-GDP) were able to induce CSF activity in vivo and in vitro. The incorporation of these compounds within appropriately designed liposomes composed of distearoylphosphatidylcholine and phosphatidylserine (DSPC/PS) increased their ability to induce CSF activity in vivo but completely abrogated their ability to induce CSF activity in vitro. Furthermore, the phospholipid composition of liposomes influenced the efficacy of glycopeptide liposomal incorporation. Thus, the serum CSF-inducing effect of MDP-GDP was considerably enhanced by incorporation of this compound within liposomes composed of DSPC/PS at a molar ratio 7:0.3 but was not modified if the DSPC/PS molar ratio was 7:3. The lipophilic derivative of MDP (D-D), MDP (D-D)-GDP, was unable to induce CSF activity in vivo or in vitro but surprisingly became active in vivo after entrapment within DSPC/PS liposomes (molar ratio 7:0.3). Our results show that appropriate liposomes may be suitable carriers to deliver CSF activity-inducing agents to macrophages in vivo.

Production of tumor necrosis factor in nude mice by muramyl peptides associated with bacterial vaccines.

Gram-negative vaccines can elicit the production of tumor necrosis factor (TNF) in mice primed by muramyl dipeptide (MDP) or by its lipophilic derivative MDP-dipalmitoyl glycerol (MDP-GDP). In mice pretreated with MDP and particularly with MDP-GDP, Bordetella pertussis vaccine was shown to be more effective than typhoid vaccine. The time course of TNF production in the blood did not indicate any difference between the effect of MDP or of MDP-GDP. In both cases the cytotoxic activity reached maximal levels by 2 h after injection of the bacterial preparations and returned to normal values between 3 and 5 h after the challenge. In nude mice, high titers of circulating TNF were also produced by combined treatment with MDP-GDP and bacterial vaccine. Moreover, in tumor-bearing mice the association of MDP or of MDP-GDP to a bacterial vaccine induced a strong hemorrhagic necrosis, whereas each treatment alone was inactive. It was also found that mice were less sick when they were primed with MDP-GDP than with MDP, and when TNF was elicited by B. pertussis instead of lipopolysaccharide. Moreover, nude mice appeared more resistant to shock and to hemoconcentration than normal mice.

Activation of alveolar macrophage tumoricidal activity and eradication of experimental metastases by freeze-dried liposomes containing a new lipophilic muramyl dipeptide derivative.

The ability of a member of a new class of lipophilic muramyl dipeptide (MDP) derivative, muramyl dipeptide-glyceryldipalmitate (MDP-GDP), to induce alveolar macrophage cytotoxic activity in vitro towards B16 melanoma cells when incorporated into two types of liposome was studied. MDP-GDP incorporated into conventionally prepared liposomes formulated from distearoylphosphatidylcholine and phosphatidylserine (7:3 molar ratio) was 10-fold more effective than liposomes containing MDP, and 7000-fold more effective than free MDP in inducing macrophage cytotoxic activity. MDP-GDP incorporated into freeze-dried liposomes was 50,000- to 100,000-fold more effective than free MDP in inducing such activity. Freeze-dried liposomes containing MDP-GDP were efficiently localized in the lungs of normal mice, and induced cytotoxic activity in the alveolar macrophages. Such liposomes were able to significantly reduce the pulmonary metastatic burden of mice carrying the B16 melanoma. These data provide evidence that this class of lipophilic MDP derivative, when incorporated into freeze-dried liposomes, is a potent inducer of macrophage cytotoxic activity in vitro and in situ, and has antitumor activity in vivo. In addition, the use of a freeze-drying procedure allows the preparation and long-term storage of reproducible liposome formulations.

Future prospects for vaccine adjuvants.

Since the landmark experiments of Ramon 60 years ago, attempts have been made to augment the humoral and cellular responses to administered antigens in order to develop more potent and less toxic vaccines. The need for an acceptable adjuvant suitable for clinical use has been underscored by recent advances in recombinant biotechnology and synthetic chemistry which have made it possible to create antigens that are smaller and better characterized, yet less immunogenic, than before. It is likely that these antigens will require an adjuvant to achieve protective immunity. Some of these same technological advances, together with a better understanding of the immune system in general, have permitted the study of adjuvants to evolve from an empirical field to a developmental one. This article discusses the currently known agents capable of immunopotentiation and possible strategies for their use in future vaccines.

Changes in rabbit febrile responses to muramyl dipeptide (MDP) after coupling to a synthetic carrier.

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.

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 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.

Priming effect of muramyl peptides for induction by lipopolysaccharide of tumor necrosis factor production in mice.

Lipopolysaccharide-induced necrosis of grafted tumors was potentiated by several hydrophilic and lipophilic muramyl dipeptide (MDP) derivatives administered a few hours prior to small amounts of lipopolysaccharide (LPS) in spite of low titers of induced circulating tumor necrosis factor (TNF). However, pretreatment with MDP derivatives did increase the level of TNF in the blood of mice challenged by a greater dose of LPS. The TNF amount in 2 h postendotoxin mouse serum reached a peak when the glycopeptide had been given 6 h before the challenge, being approximately 100-fold above that obtained in unprimed mice. The cytotoxic activity in mouse serum was inhibited by rabbit antibodies raised against recombinant mouse TNF. Although there exists a toxic synergism between BCG or MDP and endotoxin, the effect of certain MDP derivatives was not related to an increased susceptibility to the toxicity of LPS.

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.

Covalent linkage of the synthetic adjuvant MDP to the synthetic polypeptide (T,G)-A-L changes the specificity of the immune response at the T and B cell level.

It is now well established that the synthetic molecule MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) can be a good adjuvant of immunity when covalently linked to antigen. The question raised in this work is whether conjugation of antigen to the immunomodulatory molecule MDP can modify the specificity of the antibodies and T cells induced following immunization. Using the well characterized synthetic polypeptide antigen, poly(L-Tyr,L-Glu)-poly(DL-Ala)--poly(L-Lys) [(T,G)-A--L], we show that immunization of C57B1/6 (H-2b) mice with MDP-(T,G)-A--L conjugate elicits at least two types of antibody directed against the poly(DL-Ala)--poly(L-Lys) (A--L) part of the antigen, and against new determinant(s) formed by MDP and a portion of the (T,G)-A--L molecule. Interestingly, the poly-(L-Try L-Glu) side chains thought to constitute the major antigenic determinants of the (T,G)-A--L molecule were not recognized. Lymph node cells from (T,G)-A--L immunized mice can be equally well stimulated in vitro by (T,G)-A--L or by MDP-(T,G)-A--L, whereas lymph node cells from MDP-(T,G)-A--L primed animals can be stimulated only when challenged by the conjugate used for immunization, and not by the free synthetic polypeptide (T,G)-A--L. The data presented here show that the coupling of a low mol. wt molecule such as MDP (mol. wt approx. 500) to an antigen can greatly modify the immune response directed against this antigen. Furthermore, (1) different antibody specificities are elicited depending upon whether the priming is done with free MDP and antigen or with MDP covalently linked to the antigen; (2) although still accessible on the conjugate, an epitope which represents the major antigenic determinant on the free polypeptide appears to be silent when presented on the conjugate; and (3) new determinant(s) formed by the chemical linkage of the polypeptide to the synthetic adjuvant are involved in the priming of T cells.

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.

Influence of helical organization on immunogenicity and antigenicity of synthetic peptides.

Using cooperative effects of different peptide structures synthesized in tandem, we have induced an alpha-helical structure in water solution on a peptide which, alone, is unorganized. This structure is particularly relevant in this case as the selected model protein (type 24 M protein of Streptococcus pyogenes) is an extended coiled-coil system. We were thus able to assess the importance of organization or unorganization of a unique amino acid sequence with regards to its immunogenicity and antigenicity. Although in a classical manner, antibodies cross-reacting with the protein can be obtained with the short, unorganized peptide, we demonstrate that conformation-specific antibodies are raised when longer, organized peptides are used as immunogens.

Monoclonal antibodies to the synthetic adjuvant muramyl dipeptide: characterization of the specificity.

Monoclonal antibodies to MDP were prepared by hybridization of NSO myeloma cells with spleen cells of BALB/c mice immunized with MDP conjugated to methyl-BSA. Hybridomas secreting anti-MDP antibodies were selected by the binding activity of their supernates to MDP-A--L using a radioimmunoassay. After cloning in soft agar, the specificities of monoclonal anti-MDP antibodies were assayed by an inhibition of ELISA with various derivatives of MDP. Fine structural analysis of specificity for one such clone (2-4) is reported. This antibody recognizes the N-acetyl-muramic acid (N-Ac-Mur) linked to the dipeptide but not N-Ac-Mur or/and dipeptide alone. The N-Ac group on muramic acid is an important antigenic determinant and the glycopeptide linkage seems to be crucial in presenting the sugar moiety. Conservative substitution of L-Ala (i.e. by L-Ser or L-Val) had no effect on the binding ability to the antibody whereas a radical change, i.e. replacement of L-Ala by L-Pro or N-methyl-L-Ala completely abolished the antigenicity of the molecule. There was no clear correlation between biological activities of various derivatives of MDP and their ability to react with this antibody. Some possible hypotheses explaining this lack of correlation are presented.

Stimulation of human endothelial cells by synthetic muramyl peptides: production of colony-stimulating activity (CSA).

The in vivo induction of colony-stimulating activity (CSA) by well-defined immunomodulatory synthetic muramyl peptides has been demonstrated recently in mice. In the present study, we tested the capacities of three muramyl peptides to induce CSA production in human endothelial cell (HEC) cultures. Two adjuvant-active peptides (MDP and Murabutide) induced CSA in the supernatant of cultured endothelial cells, whereas an adjuvant-inactive compound had no effect. This effect of MDP and Murabutide appeared to be time and concentration dependent and was not secondary to decreased production of inhibitors of colony formation. CSA secretion by stimulated HEC required de novo protein synthesis and did not result from the release of preformed active CSA. Maximal concentration appeared in the supernatant media within the first 24 h after addition of muramyl peptides, and a substantial second CSA secretion could be observed after a subsequent 24 h reexposure. This CAS was not dialyzable and promoted granulocyte-macrophage formation of nonadherent human marrow and unfractionated murine marrow. Our data demonstrate that the human endothelial cell is a target cell for MDP and Murabutide and suggest that in vivo endothelium might play an active role in muramyl peptide-induced modulation of hematopoiesis.

Production of differentiation-stimulating factor for murine leukemic myeloblast line by monocytic cells stimulated by a nonpyrogenic muramyl dipeptide derivative.

Muramyl dipeptide (MDP) and adjuvant-active derivatives were confirmed as unable directly to induce differentiation of mouse myeloid leukemia M1 cells. They were, however, found effective in stimulating either rabbit macrophages or human blood monocytes to produce differentiation-stimulating activity (D factor). The various conditioned media (CM) thus obtained were able to induce differentiation of the myeloblastic M1 cell line as indicated by the appearance of Fc receptors and inhibition of cell proliferation. Among the synthetic glycopeptides inducing the production of D factor, murabutide (MDP[Gln]-OnBu) was as effective as MDP, although it did not stimulate monocytes to simultaneously release endogenous pyrogen. The absence of pyrogenicity in murabutide CM was attested by IV or intracerebroventricular administration to rabbits. However, in the same CM, LAF(IL1) activity estimated by potentiation of the in vitro proliferative response to phytohemagglutinin of mouse thymocytes was usually higher than that induced by MDP.

Enhancement in vivo of the antiinflammatory and antitumor activities of type I interferon by association with the synthetic immunomodulator murabutide.

The therapeutic efficacy of type I interferon (IFN) has been reported to vary considerably in different indications. The use of the cytokine as adjuvant therapy has been suggested to enhance its efficacy and reduce the toxicity frequently associated with long-term and high-dose administration. In this study, we have assessed the activity of type I IFN in the protection against and treatment of acute hepatitis induced in mice by the administration of concanavalin-A (ConA). At the same time, we have evaluated the efficacy of the synthetic immunomodulator murabutide when administered alone or in combination with type I IFN to protect against ConA hepatitis and in the treatment of tumors in MethA sarcoma-bearing mice. Our results demonstrate a prophylactic effect as well therapeutic effects of type I IFN and of murabutide in the inflammation-mediated model of liver damage. The use of combination therapy presented enhanced efficacy in inhibiting the ConA-induced elevation of plasma transaminases. Both compounds were found to suppress IFN-gamma mRNA accumulation in the livers of ConA treated mice. This activity is discussed with respect to the mechanism of action of the two immunomodulators. In addition, the combination of murabutide with type I IFN exhibited synergistic antitumor activity that was clearly seen in the significant regression of MethA tumors and resulted in almost 50 percent tumor-free mice. The potential clinical application of combination therapies using a cytokine and a safe immunomodulator is analyzed in terms of enhancing the cytokine efficacy and extending its use to new indications.

Synergistic effects between recombinant interleukin-2 and the synthetic immunomodulator murabutide: selective enhancement of cytokine release and potentiation of antitumor activity.

The use of interleukin-2 (IL-2) in the treatment of cancer has shown limited efficacy and dose-limiting toxicity. Combination therapy with other cytokines and/or chemotherapeutic agents has been attempted to enhance the antitumor activity and to reduce the effective therapeutic dose of IL-2. We recently showed, in vitro and in vivo, a synergistic activity between the synthetic immunomodulator murabutide, which is in clinical stage of development, and another therapeutic cytokine, interferon-alpha (IFN-alpha). The present study was performed to assess a possible potentiation of the biologic activities of IL-2 by its association with murabutide. Human PBMC stimulated in vitro with IL-2 and murabutide showed synergistic levels of induced mRNA accumulation and protein secretion for IFN-gamma, IL-12, and colony-stimulating factors (CSFs). No such effects were obtained on the induction of most inflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha). Furthermore, the combined administration of murabutide with IL-2 into Meth-A sarcoma-bearing mice resulted in a very significant tumor inhibition as well as in complete tumor regression in nearly 70% of the treated mice. Under the same conditions, treatment with either compound separately had little or no antitumor effect. These preclinical findings will be pursued by the evaluation of the clinical tolerance and biologic activity of the murabutide/IL-2 combination therapy in cancer patients.