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SUMMARY Activation of the cellular transcription factor nuclear factor-κB (NF-κB) 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-κB 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-κB in human and mouse cell lines. In this report, we demonstrate selective activation of NF-κB 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 (RO1) since pretreatment of cells with antioxidants blocked subsequent activation of NF-κB. However, among all the molecules tested only one lipophilic, non-pyrogenic adjuvant active muramyl peptide showed a complete lack of NF-κB activation in all cell lines tested. This molecule could well become the adjuvant of choice in future AIDS vaccines.

Mice are quite resistant to LPS toxicity but even a small dose induced a monophasic production of circulating TNF. In BCG-treated mice challenged with LPS, the greater susceptibility was associated with the capacity of producing elevated levels of TNF in the blood. During pregnancy, after adrenalectomy, and particularly after treatment with galactosamine, smaller amounts of LPS were lethal in mice. Using adrenalectomized mice, which are less sensitive to LPS toxicity than galactosamine-treated mice, it was shown that smaller doses of LPS were effective in inducing TNF release in comparison with intact animals, and that larger concentrations of serum TNF were obtained. Pretreatment of adrenalectomized mice with MDP before LPS elicited a priming effect for an enhanced TNF production that reached levels comparable to that found in BCG-primed mice. Whatever was the yield of circulating TNF, the pattern of response was similar peaking at 1.5 to 2 h to LPS injection and returning to baseline values within 4 h. Prior administration of glucocorticoid was effective in preventing the release of serum TNF in adrenalectomized mice. The level and the kinetics of serum TNF following LPS injection were not modified in pregnant or in galactosamine-treated mice, and as in control animals glucocorticoid administration prior to LPS inhibited the TNF response.

Murabutide, which belongs to the immunomodulator family of muramyl peptides, was applied directly to fresh human blood to evaluate changes in leukocyte properties. After blood incubation with murabutide, lymphocytes presented a higher responsiveness to T-mitogens, and monocytes and polymorphonuclear cells exhibited an increase in their capacity to produce hydrogen peroxide. In addition, murabutide treatment enhanced phagocytic activity of neutrophils, whereas monocytes presented a decrease in this activity. Some surface markers were also investigated in the distinct leukocyte populations, After incubation with murabutide, a larger number of lymphocytes expressed Ta1 antigen (CD W26) and transferrin receptor (CD 71). In contrast, expression of interleukin-2 receptor (CD 25) was slightly decreased. Monocytes from treated blood displayed a larger number of receptors for C3bi (CD 11b), whereas the surface marker CD 14 and the class I receptor for the Fc portion of IgG were down-regulated. Activation of polymorphonuclear cells by murabutide was confirmed by the up-regulation of the C3bi receptor, Fc receptor, and CD 14 surface antigen. The effects of murabutide on leukocytes described in this paper may contribute to understanding mechanisms of the modulating activity of muramyl peptides on specific and nonspecific immunity.

We previously demonstrated that a single intradermal injection of 10 9 irradiation-killed M. vaccae , given 1 month after starting chemotherapy, caused significant changes in responses to mycobacterial antigens. Amongst 38 patients with pulmonary tuberculosis, 29% had lymphocytes responding to common mycobacterial antigens after the injection, compared with only 11 % of 49 similar patients after an injection of saline (p To increase the proportion of responders to these antigens, six modifications of the potentially immunotherapeutic injection, randomized with injections of saline, have been assessed by biochemical, clinical, haematological, immunological and radiological criteria. Subsequent lymphocyte proliferation to mycobacterial antigens enabled the modifications to be ranked in order of efficacy. Tuberculin plus murabutide plus 10 9 irradiated M. vaccae (36% of 25), an autoclaved preparation of 10 9 M. vaccae (45% of 22), and 2 x 10 9 irradiated M. vaccae (75% of 12) were the most effective. Antibody responses in several IgG subclasses to mycobacteria, but not streptococci, were also significantly increased by the most effective modifications over the 8 weeks following injection. Detailed radiological study showed that use of the autoclaved bacilli was followed by a delay in clearing of consolidation, but by better closing of cavities than was found in the control group, suggesting enhanced, or altered, immunological activity around the lesions.

Colostrum-deprived lambs and CF1 mice were vaccinated with water-in-oil emulsion vaccines containing nonviable whole cells (WC) of Corynebacterium pseudotuberculosis with and without muramyl dipeptide (MDP). Efficacy of vaccines was determined from the survival of mice and lesions in lambs after IV injection of 10(4) colony-forming units of C pseudotuberculosis. In mice, protection was related to the concentration of WC in the vaccine. At 50, 100, or 150 micrograms of WC, protection was good (78.8%). At 10 or 25 micrograms of WC, protection was considerably less (54.7%). At high WC concentrations, protection could only be moderately increased to 82.3% with high (50 and 100 micrograms) concentrations of MDP or increased to 90% protection with low (5 and 10 micrograms) concentrations of MDP. At low WC concentrations, protection significantly decreased to 32% (P less than 0.025) with high concentrations of MDP, but significantly increased to 72.5% (P less than 0.025) with low concentrations of MDP. Therefore, the amount of protection with lower concentrations of WC and MDP was comparable with the amount of protection with higher concentrations of WC without MDP. In lambs, high prechallenge antibody titers (geometric mean titers from 5.1 to 5.4 by day 35) were observed after vaccination with WC. Protection and vaccination site abscesses in lambs were related to the concentration of WC and MDP. Pulmonary or vaccination site abscesses were not observed in 4 of 4 lambs vaccinated with 1 mg of WC + 50 micrograms of MDP.

Azidothymidine (AZT) is the only drug currently approved for treating HIV infections. AZT treatment results in decreased mortality and frequency of opportunistic infections but is associated with bone marrow depression or failure (Pizzo 1988). The termination of AZT treatment results in increased levels of viral core protein p24 and decreased numbers of CD4+ cells (Jackson 1988).

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.

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.

Contractions evoked by muramyl dipeptide (MDP), a synthetic compound possessing immunostimulatory properties, were studied in several isolated nerve-smooth muscle preparations. The contractions by micromolar concentrations of MDP were evoked either by direct interaction with smooth muscle (rat stomach strip) or at least partly indirectly via neurogenic stimulation (guinea pig ileum); the effect was stereospecific since the MDP-D was not active. The insensitivity of the preparations to serotonin (5-HT), either inherent (vas deferens) or after 5-HT antagonists or after desensitization to 5-HT, prevented or markedly reduced the contractile activity by MDP. On the other hand, a nanomolar concentration of MDP enhanced the sensitivity of the rat stomach strip to 5-HT.

Injection of MDP into mice induces a rapid elevation of monocyte-macrophage CSA in the serum. This effect can also be observed in LPS-hyporesponsive C3H/HeJ mice. MDP and LPS induce CSA synergistically in normal mice. In contrast to the tolerance that is rapidly observed after repeated administration of LPS, MDP does not lose its capacity of inducing serum CSA after repeated injections. Repeated daily injections of MDP also fail to induce tolerance to the LPS-CSA inducing effect. Furthermore, whereas mice rendered tolerant to LPS become hyporesponsive to many other bacteria or bacterial products, they remain responsive to MDP. These data showing that MDP can act synergistically with another CSA inducer, can be injected repeatedly, and can stimulate mice unresponsive to LPS suggest potentially important in vivo applications.

In the present study, well-defined immunomodulatory synthetic glycopeptides were used to investigate putative regulatory mechanisms of in vitro IL 2 production by normal human peripheral blood mononuclear cells. MDP (muramyl dipeptide) and two of its structural analogs, murabutide and MDP-DD, were shown to inhibit the in vitro PHA-induced IL 2 production in a majority of normal individuals tested. Involvement of prostaglandins in such an inhibitory effect was suggested by the fact that indomethacin completely abrogated the MDP-induced suppression. There was, however, some evidence indicating that the inhibition induced by the synthetic glycopeptides and that induced by PGE2 were somewhat different. Indeed, although the PGE2-induced suppression of IL 2 production was completely reversed by preirradiation of PBMNC, this was not observed for the MDP-dependent inhibition. In addition, PMA was able to abrogate the suppression induced by MDP, whereas it increased that of PGE2. From these data we propose that at least two independent pathways in the regulation of human IL 2 production exist: a one-signal pathway already described in which PGE2 directly triggers a radiosensitive suppressor T cell subset; and a second pathway with two signals, one given by PGE2 and a second one given by agents such as muramyl peptides. These two signals are required to activate a radioresistant suppressor cell subset.

Leishmania tropica causes a lethal visceral disease in highly susceptible BALB/c mice, with many immunopathologic features resembling those in human kala-azar. The responses to thymus-independent antigens of Type 1 and 2 (TI-1, TI-2) were compared in infected mice of susceptible BALB/c and resistant C57BL/6 strains at various times after infection. The infected BALB/c mice had an augmented response to both types of antigens at 45 days after infection. Later (day 76), the response to trinitrophenylated lipopolysaccharide (TNP-LPS, a TI-1 antigen) was diminished but that to dinitrophenylated Ficoll (DNP-Ficoll, a TI-2 antigen) remained statistically above the response of uninfected mice. The response of the resistant strain to either antigen was not modified as a result of the infection. Both strains showed significant polyclonal activation, which was considerably greater in the BALB/c than in the C57BL/6 mice. The observations presented here are in contrast to the widely held belief that a generalized nonspecific immunosuppression occurs in L. tropica infected BALB/c mice.

Two purified peptidoglycans 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.

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-mediated 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.

Adjuvant muramyl dipeptides enhanced the intracellular oxidative burst induced by phorbol myristate acetate in purified human polymorphonuclear (PMN) cells and in monocytes. A stronger priming effect was obtained when muramyl dipeptide was conjugated to a protein carrier. Recombinant human tumor necrosis factor (TNF) did not modify the level of intracellular NBT reduction in PMA-stimulated PMN, although it slightly increased the secretion of superoxide anion. In contrast, TNF enhanced the intracellular oxidative burst of monocytes even at the concentration of 10 pg/ml. In human PMN only, the combination of TNF and muramyl dipeptide induced a higher oxidative response than each stimulant alone.

Non-specific immunostimulants were used in an attempt to protect baboons from infection by schistosomes. Subcutaneous vaccination with cord factor (4·50 mg) and muramyl dipeptide (4·56 mg) 6 days before percutaneous exposure to 3000Schistosoma haematobiumcercariae/baboon (c.p.b.) failed to protect naive baboons: baboons with a 7-month-old, 5000 c.p.b.S. haenzatobiumprimary infection had developed too strong a natural immunity to detect any protection attributable to vaccination. Subcutaneous vaccination with 0·4 ml of Bacillus Calmette-Guerin (BOG, 1–8 x 108colony forming units7sol;ml) 4 days before exposure to 1000 c.p.b.S. mansonigave a significant (38%) reduction in worm load compared with controls. However, vaccination with 0·8 (intramuscular) and 0·2 (intradermal) ml of BOG 11 days before exposure toS. mansoni800 c.p.b. did not protect naive baboons, nor did it significantly reduce challenge worm recovery from baboons with a 13-week-old, 500 c.p.b.S. mansoniprimary infection. Obvious pathology was seen at the site of vaccination in the first but not the second BCG experiment. These results partly support the findings in mice that non-specific macrophage and monocyte activators give partial protection against schistosome infections but they also illustrate that rodents and primates do not necessarily react identically. Hence, findings from rodent models should be extrapolated to man with some caution.

Synthetic antigens have been shown, in experimental models, to induce protective immunity against a variety of pathogens. These studies have demonstrated that, due to their low immunogenicity, these synthetic antigens required conjugation to carrier molecules. Therefore, the choice of appropriate carriers for human immunization by future synthetic vaccines is a major issue. Tetanus toxoid is generally considered to be an effective potential carrier devoid of side-effects. However, the present study performed in mice with two synthetic vaccine models demonstrates that the immune response against the synthetic epitopes conjugated to tetanus toxoid can be suppressed by pre-existing immunity against this same carrier. Because most humans have been exposed to this antigen, this effect may have important implications for the development of synthetic vaccines.

A water-soluble extract of Nocardia opaca (NWSE) induced a very strong stimulation of rabbit and mouse spleen lymphocytes and a weak, but significant, increase of thymidine incorporation by human peripheral lymphocytes and monkey blood, or spleen cells. As was demonstrated by the migration inhibition factor assay, the strong mitogenic activity observed with mice spleen cells was not related to a state of natural impregnation. In the same experiments, the various lymphocytes were also incubated with M. smegmatis water-soluble adjuvant (WSA) and S. enteritidis lipopolysaccharide (LPS). No mitogenic activity was shown with WSA even when this extract was incubated with the lymphocytes of human tuberculin responders, showing that this preparation is free of tuberculoprotein contaminants. Finally, it must be noted that, contrary to what is observed in mice, LPS did not induce thymidine incorporation by monkey or rabbit spleen cells.

Two muramyl dipeptides, N-acetylmuramyl-L-alanyl-D-isoglutamine and its adjuvant-inactive isomer N-acetylmuramyl-D-alanyl-D-isoglutamine, were examined for their ability to protect mice carrying the CBA/N immune deficiency gene (xid) against lethal bacterial challenge. Prophylactic treatment with N-acetylmuramyl-L-alanyl-d-isoglutamine gave significant protection against Streptococcus pneumoniae, Salmonella typhimurium, and Salmonella enteritidis infection. N-Acetylmuramyl-D-alanyl-D-isoglutamine was unable to confer protection. Incorporation of the lipophilic glycerol dipalmitate derivatives of the two muramyl dipeptides within liposomal carriers resulted in a significant enhancement of anti-infectious activity, both with respect to number of survivors and length of survival. Liposomal muramyl dipeptides were 10- to 15-fold more potent than free muramyl dipeptide; enhanced potency was most evident with N-acetylmuramyl-D-alanyl-D-isoglutamine. Prophylactic treatment with liposomes containing the lipophilic muramyl dipeptides resulted in enhanced clearance of bacteria from the blood (greater than 3-fold increase in rate) when compared with that of hydrosoluble N-acetylmuramyl-L-alanyl-D-isoglutamine, indicating a correlation between reticuloendothelial stimulation and anti-infectious activity.

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.

Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti-RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.

Resistance to schistosomiasis in mice can be acquired either specifically, by primary infection with Schistosoma mansoni, or nonspecifically, by treatment with a variety of unrelated agents such as bacille Calmette-Guérin. Several immunoadjuvants related to mycobacteria were examined for their ability to induce resistance to schistosomiasis. Natural cord factor (6,6'-trehalose dimycolate), a 100-carbon synthetic cord factor analogue, and dipalmitate trehalose induced significant protection. Trehalose dibehenate and muramyl dipeptide did not induce consistent protection. Since protection acquired by primary schistosomal infection or by any of these potentiating agents is partial, their possible additive effect was evaluated. The resistance of mice with schistosomiasis that were injected with trehalose dipalmitate and challenged with schistosomal cercariae was increased, as assayed by recovery of schistosomula from the lungs and of adult worms from the portal system. Thus, these synthetic adjuvants not only induce partial protection against schistosomiasis, but also significantly enhance acquired immunity in mice with primary infections.

The effect of human recombinant tumor necrosis factor (TNF) was studied in vitro on human endothelial cells. TNF (1-1000 pg/ml) induced a dose-dependent increase in PAI level in the supernatant from 6 to 25 U/ml as estimated against urokinase. This effect was time-dependent. It was not suppressed by Polymyxin B thus excluding a possible contribution of an endotoxin contamination. Fibrinoenzymography performed after SDS-PAGE showed that this inhibitor neutralized urokinase and tissue plasminogen activator and gave rise to high molecular weight complexes. TNF (30 micrograms/kg) was also injected in rat. Blood fibrinolytic activity determined 4 hr later was decreased as estimated by the prolongation of the euglobulin clot lysis time from 37 to 188 min. Fibrinoenzymographic profile of the plasma was then characterized by a fainting of the tPA lysis band but the capacity of plasma to neutralize urokinase was not significantly modified. These results suggest that TNF could alter the fibrinolytic balance by stimulating PAI production at the endothelial level. This might be of importance in synergy with the TNF-induced procoagulant activity for promoting vascular occlusion of tumor capillaries.

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 nonimmunogenic MDP analogs possessing immunoadjuvant activity.

In a previous study we demonstrated that lipopolysaccharide failed to elicit nonspecific resistance in C3H/He lipopolysaccharide low-responder mice against Klebsiella infection in contrast to its activity in a closely related histocompatible high-responder subline, C3HeB/Fe. Complete restoration of lipopolysaccharide-induced protection against 10(5) Klebsiella was obtained in the present study by transferring bone marrow from high-responder mice to the highly deficient C3H/He mice. The ability of C3H/He mice to clear and destroy bacteria in 5 h was also transferred by C3HeB/Fe marrow cells. In contrast, when high-responder C3HeB/Fe mice were reconstituted with low-responder bone marrow, the clearance and destruction of K. pneumoniae were similar to what is observed in the high-responder strain, but survival was only temporary. Collectively, our data show that the failure of C3H/He mice to respond to lipopolysaccharide with nonspecific immunity is due to a defect in two types of bone-marrow-derived cells--radioresistant and radiosensitive.

A synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), was previously shown to enhance polyclonal antibody response in murine spleen cell cultures. When MDP was added to the culture together with a potent murine B cell mitogen (such as bacterial lipopolysaccharide (LPS)), it inhibited completely the LPS-induced polyclonal activation without affecting either the 3H-thymidine incorporation or the number of blast cells in cultures. Strong suppression of mitogen-induced polyclonal activation by MDP was obtained by using a large range of cell concentrations in cultures and over various dosage levels of the stimulating mitogens (LPS and NWSM). An inhibition could be obtained even when MDP was added 24 hr after the addition of the mitogen, and highly significant suppression was observed in the absence of cell division in the cultures.

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.

Antibodies were raised in mice immunized with several recombinant and synthetic peptides of the circumsporozoite protein of Plasmodium falciparum. The antibodies were evaluated for protective activity in a human hepatocyte culture system. They exerted their protective effect against the parasite at three points: sporozoite attachment to the hepatocyte surface, entry, and subsequent intracellular development. Inhibition of attachment and entry were found to be related to the antibody titer against the authentic circumsporozoite protein on the sporozoite surface, especially when peptides were administered with alum or complete Freund's adjuvant. Even when invasion was not totally inhibited, the presence of abnormal trophozoites and a frequent inhibition of schizont development in long-term cultures suggested continued activity of antibodies at the intracellular level after sporozoite penetration had been completed.

Digestion by lysozyme of delipidated cells of Mycobacterium smegmatis liberates a water-soluble immunoadjuvant fraction which is chemically very similar to the water-soluble adjuvant (WSA) obtained previously from purified cell walls, but which contains somewhat more non-peptidoglycan amino acids. The yield of peptidoglycan-arabinogalactan complex is about 10 times greater starting from whole cells than from cell walls. The main biological properties of this “neo-WSA” are described: it increases circulating antibodies to ovalbumin in guinea pigs, it does not produce polyarthritis in rats or induce hypersensitivity to tuberculin, it does not increase susceptibility to histamine or hyperreactivity to endotoxin, and does not produce spleen and liver hypertrophy. Analogous immunostimulant fractions have also been obtained from delipidated cells of Nocardia opaca by lysozyme treatment.

Summary The bactericidal effect of normal mouse serum, generally ascribed to natural “O” antibodies, can be removed by absorption with rough microorganisms. Passive hemagglutination tests disclose the presence of thermolabile, non-agglutinating “R” antibodies in normal mouse serum; titers in germ-free animals are much lower. Horse hyperimmune serum against rough S. typhimurium protects mice against infection with unrelated virulent smooth K. pneumoniae. Provided the Klebsiellae are preincubated in normal mouse serum bactericidal activity can also be demonstrated in vitro. This activity does not require the presence of complement and can be removed either by the same Klebsiella strain or by rough strains of Salmonellae but not by the smooth strain of Salmonellae from which these mutants originate. Although these effects would most easily be accounted for by specific antibodies it is also possible that they are instead attributable to a serum protein capable of charge interaction with rough antigens. In any event these findings, together with previous observations concerning degradation of somatic antigen and phenotypic modification of Klebsiellae recovered from endotoxin-treated mice, have led to the development of a new concept of natural immunity to Enterobacteriaceae. In this hypothesis, enzymatic factors capable of attacking cell wall components unmask R antigenic sites common to many bacterial strains and species. Thereafter the presence of a few types of “R” antibodies or of serum factors reacting with rough antigens have the capability of coping, like masterkeys, with a very wide range of infections due to serologically unrelated organisms.

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 haptenprimed 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.

The circumsporozoite (CS) protein of P. falciparum contains an immunodominant epitope, NADP, that is repeated 37 times in the native molecule. The presence of proline in the coat proteins of the Plasmodium parasite at various developmental stages and strains is a frequent occurrence. In this study we evaluate the influence of substitution of proline residues by glycine on the immunogenic behavior of two tandemly repeated peptides linked via glutaraldehyde to a protein carrier: The (NANP)4 P. falciparum circumsporozoite peptide and its glycine-substitute analog, (NANG)4. The results obtained show that the (NANP)4 induces antibodies which recognize the peptide free in solution, bound on a solid phase, and linked to a carrier protein. It has been previously reported that such antibodies recognize the antigenic sites of the peptide in the native protein on the surface of the sporozoite. Antibodies raised against (NANG)4 in the same experimental conditions as (NANP)4, cannot recognize the peptide free in solution or bound to the solid phase. However, these antibodies can react with the peptide when it is linked to a protein carrier. The coupling of a glycine-containing analog to a carrier results in a significant shift in its conformation, allowing it to be recognized by the antibodies.

Synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine, also called MDP for muramyl dipeptide, is a copy of a fragment of bacterial peptidoglycan. MDP and several of its derivatives have marked immunological and pharmacological activities. Besides being adjuvants and capable of producing hyperthermia, they can also induce an increase of slow-wave sleep (SWS). It became apparent in the course of the investigations that in many cases the effects produced by MDP could be mediated by the macrophages. Therefore, after briefly reviewing their influence as macrophage activators, we will focus on certain newer findings such as the somnogenic activity of MDP and of monokine.

MDP is a synthetic copy of part of the bacterial cell wall which has been shown to be the minimal structure capable of replacing Mycobacteria in Complete Freund Adjuvant. Several hundred analogs of this molecule have been produced and have been studied in various immunopharmacological systems. Special emphasis will be given here to their pyrogenic and somnogenic effects and to certain similarities which exist between MDP and Interleukin-1.

A monoclonal anti-MDP antibody was found to bind to "Slow Wave Sleep" factor. This result confirms that this factor is a muramyl peptide and furthermore shows that it contains a structure characteristic of the synthetic adjuvant and of the bacterial cell wall, i.e. an acetylated muramic acid bound to L-alanine. This antibody was also shown to specifically inhibit a biological activity of a purified human monokine which induces fever. Because of these results and other recent observations we propose that a bacterial structure is present in certain mammalian mediators.

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.

Enhancement of nonspecific host defenses against bacterial challenge has been a long-standing goal often thwarted by variable efficacy, inconsistent bioassay, and paradoxic immunosuppression. Muramyl dipeptide provides enhanced survival after intravenous challenge with less than 8 x 10(3) Klebsiella, as well as improved local control of infection at sites of intramuscular bacterial injection, with and without a surgical foreign body. No depression of host response was seen over wide ranges of doses and intervals. Muramyl dipeptide provides a new order of efficacy and safety and warrants continued careful assessment.

The visceral and lethal infection produced in BALB/c mice by Leishmania tropica (major) is accompanied by splenomegaly, anaemia and reversal of albumin-to-globulin ratio. The percentages of both B and T cells are decreased in the spleen. The spleen and lymph nodes become populated with large Ig-, Thy 1.2- 'null' cells. The similarity of some of these parameters with those produced in human kala-azar is discussed.

The ability of three members of a new class of lipophilic muramyl dipeptide derivative to induce murine macrophage tumoricidal activity after liposomal incorporation was investigated. Liposomes containing the glycerol dipalmitate (GDP) derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-glutamine-n-butyl ester, and N-acetylmuramyl-D-alanyl-D-isoglutamine were 5000, 2000, and greater than 10,000-fold more potent than the free muramyl dipeptides in inducing peritoneal macrophage tumoricidal activity in vitro. In situ activation of peritoneal macrophage tumoricidal activity showed that liposomal muramyl dipeptide-GDP derivatives were more potent than free hydrosoluble or sonicated muramyl dipeptide-GDP preparations. In situ induction of alveolar macrophage tumoricidal activity after i.v. treatment was observed with liposomes containing muramyl dipeptide-GDP derivatives, but not with hydrosoluble or sonicated lipophilic derivatives. Liposomes containing muramyl dipeptide-GDP derivatives were therapeutically active against experimentally induced pulmonary B16 melanoma tumors in C57BL/6 mice. These results demonstrate that when incorporated within liposomes this class of lipophilic muramyl dipeptide derivative is a potent inducer of macrophage tumoricidal activity both in vitro and in situ, and possesses antitumor activity in therapeutic treatment protocols.