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The capsular polysaccharide (CP) of Pasteurella haemolytica serotype A1 is a poor immunogen for the prevention of pneumonic pasteurellosis of ruminants. To improve CP immunogenicity, vaccines were prepared with 1.0 mg CP dose-1 with and without the synthetic adjuvant, muramyl dipeptide (MDP; range 0.2-1.0 mg) or a lipophilic derivative, MDP-sn-glyceryl-dipalmitoyl (MDP-GDP; range 0.1-1.0 mg). The optimum effective concentration of adjuvant was first determined in lambs and calves and then the efficacy of CP +0.5 mg MDP and CP +1.0 mg MDP-GDP was compared with that of two commercial vaccines in calves. After immunization with CP, antibody titers in lambs and calves were typical of that seen with polysaccharide immunogens and characterized by an early IgM response followed by later IgG1 and IgG2 responses. CP + MDP or MDP-GDP vaccines induced significantly higher IgM, IgG1, and IgG2 titers. After transtracheal challenge of immunity with P. haemolytica serotype A1, extensive pulmonary consolidation containing P. haemolytica (10(6)-10(8) c.f.u. g-1) was seen in all lambs and calves vaccinated with CP alone and was not significantly different (P < 0.05) from the consolidation and concentrations of organisms in nonvaccinated challenge controls. In lambs, vaccines containing 1.0 mg CP +0.05 mg MDP or MDP-GDP significantly reduced pulmonary consolidation and concentrations of P. haemolytica in lung lesions. In calves, vaccines containing 0.2 mg MDP, 0.5 mg MDP, or 1.0 mg MDP-GDP also significantly reduced pulmonary consolidation and concentrations of P. haemolytica in lung lesions. Vaccines containing CP +0.5 mg MDP and CP +1.0 mg MDP-GDP induced high titer bactericidal antibodies by 7 days and were more efficacious than two commercial vaccines. Potentiation of CP with MDP or MDP-GDP has great promise in furthering the potential of CP as a vaccine immunogen for the prevention of pneumonic pasteurellosis.

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.

The cytolytic Clostridium perfringens delta toxin lyses selectively cells which express ganglioside GM2. In this study, we investigated whether delta toxin can be used to characterize GM2 on tumor cell membranes and as an antitumor agent. The sensitivity to lysis by delta toxin of various murine and human malignant cell lines and also normal tissues was quantified using a 51Cr-release assay. The cytotoxicity titers were correlated with the 125I-labeled toxin binding capacity of sensitive and insensitive cells. Seven of eight human melanomas tested were lysed by the toxin and, of these, four were very sensitive (cytotoxicity titers below 12 ng of toxin). All neuroblastomas, gliomas and the retinoblastoma tested were lysed with 3-18 ng of toxin. Three of six carcinomas and one of two sarcomas were also very sensitive (cytotoxicity titers 0.6-15 ng) whereas leukemias and lymphoma cells were insensitive. Normal human tissues were insensitive (erythrocytes, skin fibroblasts) or poorly sensitive (brain, lung, spleen). The in vivo antitumor activity of delta toxin was tested in tumor-bearing mice. Daily intra-tumor injections of 0.5-1 mg of toxin for 4-5 days in carcinoma Me180- and melanoma A375-bearing nude mice, and neuroblastoma C1300-bearing A/J mice significantly inhibited tumor growth for 12-36 days. Intravenous administration of 100 ng of toxin per day for 5 days in Me180-bearing nude mice and C1300-bearing A/J mice gave significant inhibition of tumor growth only during the treatment period, and 10 injections of the same dose of toxin had no significant effect on SK-MEL28, a tumor lacking GM2.

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.

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.

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 myocobacterial antigens after the injection, compared with only 11% of 49 similar patients after an injection of saline (p less than 0.03). 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.

We demonstrated recently that the production of tumor necrosis factor (TNF) is induced in normal mice and in the immunosuppressed nude mouse model by the administration of muramyl dipeptide (MDP) derivatives followed by endotoxin (lipopolysaccharide). In the present study, the ability of this treatment to induce the production of TNF in mice receiving cyclophosphamide (CY) was examined. Two days following treatment with high-dose CY (250 mg/kg), mice exhibited leukocytopenia and drastically reduced splenic weight. However, these animals remained capable of producing TNF, albeit at lower levels, when treated with MDP derivatives and lipopolysaccharide (LPS), particularly when the lipophilic analogue MDP-dipalmitoyl glycerol (GDP) was utilized. TNF was also induced by the administration of MDP-GDP and LPS to Meth A sarcoma-bearing mice treated with this dose of CY. Furthermore, in all animals receiving this combination therapy, sarcoma necrosis and complete regression were obtained without any sign of tumor regrowth. A dose of 100 mg/kg CY was not effective for inhibiting tumor regrowth under the same experimental conditions. These results demonstrated that the anti-tumor activity of endogenously induced TNF is potentiated by combined therapy with a high dose of CY.

In the search for compounds capable of inducing endogenous production of colony-stimulating factor (CSF) and possessing activity against human immunodeficiency virus (HIV), an immunomodulator, muramyl dipeptide (MDP), was investigated. MDP can enhance monocyte-macrophage CSF in serum and promote nonspecific resistance against a variety of microbial pathogens. MDP exhibited an inhibitory activity against HIV infection of CD4+ H9 lymphocytes and U937 monocytoid cells. An inhibitor of viral reverse transcriptase, 2', 3'-dideoxyadenosine, produced potent inhibition in cultures which were similarly infected with HIV. MDP could partially reduce antigen production in persistently HIV-infected KE37/1 lymphocyte cultures.

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.

Three different synthetic polyvalent vaccines have been constructed by conjugating four synthetic peptides without any carrier protein. The peptides were copy fragments of two bacterial antigens (Streptococcus pyogenes M protein and diphtheria toxin), two parasitic antigens (circumsporozoite protein of Plasmodium falciparum and Plasmodium knowlesi), and one viral antigen (hepatitis B surface antigen). Outbred guinea-pigs immunized with polyvalent vaccine containing streptococcal, diphtheric, P. knowlesi and hepatitis peptides raised high specific antibody response against the four specificities. Individual T cell analysis demonstrated that hepatitis peptide bears T dominant epitope. A similar immune response was obtained with a second polyvalent vaccine where the P. knowlesi peptide had been replaced by the P. falciparum peptide. In both experiments the malarial peptides behave like pure B epitopes. Prediction of immunodominant helper T-cell antigenic sites were performed with the five peptides using computer algorithm. Hepatitis and diphtheric peptides were selected whereas the streptococcal peptide was rejected although it can experimentally contain a T epitope. To confirm this result animals were immunized with a third polyvalent vaccine which does not contain the hepatitis peptide. No T cell proliferation or antipeptide antibodies were detected. These results demonstrate that the cooperative immune response requires a certain degree of antigenic complexity for the induction of antibody response.