A method for the preparation of adjuvant peptide mimetics of GMDP with the use of monoclonal antibodies and combinatorial libraries of peptides in the format of phage display

A method for the preparation of peptide mimetics of GMDP which could exhibit adjuvant activity without the negative effects of GMDP is described. The search for peptides with GMDP-like adjuvant activity was performed using highly specific monoclonal antibodies against GMDP and combinatorial peptide libraries in the format of phage display. Various elution methods were used for the immunoaffinity enrichment of the libraries during the course of the preparation of highly active and specific peptides. A sole peptide Arg-Val-Pro-Pro-Arg-Tyr-His-Ala-Lys-Ile-Ser-Pro-Met-Val-Asn (RN) was obtained by the elution of phage particles from the immunosorbent with a 1 μM solution of the natural ligand (GMDP). Elution with a buffer with a low pH value (0.1 M glycine-HCl, pH 2.2) gave two other peptides: Ser-Gly-Arg-Val-Ala-Val-Ser-Pro-Asp-Ser-Pro-Leu-Phe-Tyr-Pro (SP) and Arg-Tyr-Gly-Gly-Ser-Val-Leu-Asn-Ile-Glu-Cys-Gln-Phe-Tyr-Gly (RG). Affinity constants for the RN and SP peptides proved to be 3.6 × 10⁸ and 3.5 × 10⁸ M⁻¹, respectively. The specificity of the interaction with the monoclonal antibodies was checked by the competitive displacement of the peptides from the antigen-antibody complex by GMDP. The RN peptide exhibited adjuvant activity similar to that of GMDP, but had no pyrogenic effect characteristic of GMDP. The described method could be used for the search for mimetics of biologically active low-molecular compounds.     

Polyprenyl Phosphates Induce a High Humoral and Cellular Response to Immunization with Recombinant Proteins of the Replicative Complex of the Hepatitis C Virus

The search for new adjuvants remains the critical task for the creation of hepatitis C vaccines due to the weak immunogenicity of biotechnological products. When immunizing mice with the recombinant proteins NS3 and NS5B of the hepatitis C virus (HCV), the adjuvant activity of three immunomodulators was compared. Phosprenyl® on the basis of polyprenyl phosphate (PPP), chemically synthesized analogue of the bacterial cell wall glucosaminyl muramyl dipeptide (GMDP), and IFN-α recombinant protein were tested. GMDP increased the activity of IgG1 antibodies 4–6 times but did not stimulate the production of IFN-γ; IFN-α has not shown any adjuvant properties. The introduction of recombinant HCV proteins together with PPP in low doses increased the activity of IgG2a isotype antibodies 4–7 times and increased IFN-γ secretion 3 times. Thus, it was first shown that PPP polarizes the immune response to Th1-type and is a promising adjuvant for the development of a vaccine against hepatitis C.     

Comparison of adjuvant activities of glucosaminyl-muramyl dipeptide and of the gene coding for granulocyte-macrophage colony-stimulating factor in DNA immunization against herpes simplex virus

Adjuvant activities of granulocyte-macrophage colony-stimulating factor (GM-CSF) and synthetic glucosaminyl-muramyl dipeptide (GMDP) were studied in immunization against type 1 herpes simplex virus (HSV1). Gene encoding the gD HSV1 protein (pDNAgD) was used as an immunogen. Gene encoding GM-CSF in pDNAGM-CSF plasmid, which was developed for eukaryotic expression, and GM-DP were used as immune response modulators. GMDP and plasmid DNA with inserted GM-CSF gene enhanced T-cell immune response to HSV1 after a single injection (pDNAGM-CSF) or 24 h before (GMDP) immunization with the gD HSV1 gene. Both adjuvants increased protective effect of DNA-immunization by a virus gene with 63 up to 100% after injection of two genes and up to 96% after the viral gene was inoculated 24 h after GMDP. These high effects indicate that further investigation of anti-HSV1 DNA-based vaccines used with genetic and peptide adjuvant is prospective.     

Comparison of the Adjuvant Activity for the Glucosaminyl-Muramyl Dipeptide and the Granulocyte-Macrophage Colony-Stimulating Factor Gene in Gene Immunization against the Herpes Simplex Virus

The adjuvant activity in DNA immunization against herpes simplex virus type 1 (HSV1) was studied for the granulocyte-macrophage colony-stimulating factor (GM-CSF) synthesized from an eukaryotic expression plasmid (pDNAGM-CSF) and for the synthetic glucosaminyl-muramyl dipeptide (GMDP). A plasmid containing the HSV gD gene (pDNAgD) was used as an immunogen. GMDP and pDNAGM-CSF each enhanced the T-cell immune response to DNA immunization. The protective effect of DNA immunization increased from 63 to 100% when the two plasmids were injected simultaneously and to 96% when pDNAgD was injected one day after injecting GMDP. The results showed that DNA vaccines combined with genetic or peptide adjuvants are promising for DNA immunization against HSV.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 504–512.Original Russian Text Copyright © 2005 by Kozlov, Klimova, Shingarova, Boldyreva, Nekrasova, Guryanova, Andronova, Novikov, Kushch.     

A comparative study of the immunological properties of peptidoglycans of different origins

The action of peptidoglycans (PG) of different origin has been experimentally studied in vivo. In these experiments PG of bacterial origin, such as blastolysin (BL), and synthetic PG, viz. muramyldipeptide (MDP) and its analog glucosaminylmuramyldipeptide (GMDP) have been used. Their toxicity, allergenic action, their effect on the phagocytic activity of peritoneal exudate macrophages (PEM), the accumulation of antibody-producing cells in the spleen, antibody titer in the blood serum and delayed hypersensitivity to nonbacterial antigens have been determined. As revealed in this study, BL does not differ from MDP in its toxicity and allergenic action. The phagocytic activity of PEM under the influence of BL only insignificantly differs from their activity under the influence of MDP, but is lower than under the influence of GMDP. The adjuvant action of BL is somewhat higher than that of synthetic PG.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Search for ligand of N-acetylglucosaminyl-N-acetylmuramyl dipeptide using its peptide mimetic

The method for searching for ligands exerting an adjuvant effect is described. The method involves isolation of polysomes using an immobilized peptide mimetic of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) — RN-peptide. After the affinity chromatography and washing, RN-peptide complexes with the target sequences were dissociated with guanidine hydrochloride. The obtained mRNA was used for cDNA synthesis and subsequent cloning in an expression vector. Further studies showed the effectiveness of this method. Clones interacting with the peptide were selected using biotinylated RN-peptide. It was found that all clones encode a sequence identical to the protein YB-1. Recombinant antibodies against protein YB-1 were selected from a phage display human scFv library. Using these antibodies, we determined the binding constant of RN-peptide to protein YB-1. Competitive analysis showed that RN-peptide and GMDP compete for the same portion of YB-1 at molar ratio 1: 12.     

Immunostimulating properties of synthetic derivatives of muramyl dipeptide and glucosaminyl muramyl dipeptide in vitro

Production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by macrophages of the spleen and peritoneal exudate of mice as well as cytotoxic factors (CFs) by murine splenocytes after in vitro activation was estimated. All the derivatives of muramyldipeptide (MDP) and glucosaminylmuramyldipeptide (GMDP) were able to induce production of TNF and CFs. In the presence of lipopolysaccharide (LPS), the effect was always higher. The response of the spleen macrophages to the effect of the preparations was higher than that of the peritoneal ones and ++non-fractionated splenocytes. GMDP and GMDP4 especially in the presence of LPS had the highest effect on induction of IL-1 by the murine peritoneal macrophages. On the contrary, MDP induced higher IL-1 synthesis by the spleen macrophages. The most active substances with respect to production of TNF, CFs and IL-1, i.e. MDP3 and GMDP4, might be recommended for immunotherapy of syngeneic tumors in animals.     

Effect on human complement of blastolysin and the glycopeptide (MDP and GMDP) and carbohydrate fragments of peptidoglycans

Along with complement activation by the classical pathway, blastolysin, an antitumor and adjuvant preparation of Lactobacillus bulgaricus peptidoglycans, effectively inhibits the transformation of C3 in to C5 convertase. Values of inhibition maximum and dissociation constants of the reversible C3b-acceptor complex for blastolysin and main immunological active structural moieties of peptidoglycans (GMDP, MDP) and their inactive carbohydrate components (N-acetylglucosaminyl-N-acetylmuramic acid, N-acetylglucosamine, and N-acetylmuramic acid) have been determined. Immunostimulator concentrations for blastolysin, GMDP, and MDP in inhibition of the C5 convertase formation (C3b binding) correlate with their doses in vivo (animal blood), displaying antitumor activity.     

An increase in the immunogenicity of bacterial antigens under the influence of one of the derivatives of muramyl dipeptide]

As revealed in animal experiments, glucosaminylmuramyl dipeptide (GMDP), the synthetic analog of muramyl dipeptide, when introduced intraperitoneally in a single injection or orally, exhibits adjuvant activity with respect to Citrobacter 0-antigens, Shigella flexneri and enhances the protective properties of dysentery and pertussis vaccines. The stimulating properties of GMDP depend on its dose, the route of its administration, the time elapsed after its administration, its ratio to the concomitant doses of bacterial antigens and to the dose of the virulent culture used for challenge.     

Use of synthetic carriers and adjuvants for increasing the immunogenicity of a synthetic peptide from the CS-protein of Plasmodium falciparum]

In order to increase immunogenicity of the peptide (NANP)3, we have prepared a large set of fully synthetic constructions based on the peptide, glycopeptide adjuvant GMDP and some synthetic carriers. Immunogenicity of these constructions was tested on mice (line C57B1/6) responding to the peptide polymer (NANP)40 without carrier and on mice (line BALB/c) not responding to this antigen. Immunogenic constructions based on synthetic polytuftsin induced as high titres of anti-(NANP)3 antibodies as the standard conjugate KLH--(NANP)3. The chimeric peptide consisting of (NANP)3 and tuftsin dimer induced anti-(NANP)3 antibodies in both lines of mice as well. The GMDP covalent attachment to the immunogenic constructions increased the antipeptide antibodies titre. The results are discussed in terms of an approach to synthetic vaccines.     

Inhibition of systemic TNF-alpha cytotoxicity in cancer patients by D-peptidoglycan

The current study was designed to investigate direct inhibitory effects of N-acetylglucosaminyl-muramyldipeptide (GMDP) over the cytotoxic nature of TNF-α. A lactate dehydrogenase (LDH) assay of the inhibition of TNF-α cytotoxicity was donein vitro on the following cell lines: A549 (human lung carcinoma cells), A431 (human breast cancer cells) and L929 (mouse breast cancer cells). In a double-blind placebo-controlled trial, cancer patients with an elevated activity of all five LDH isoensymes were randomized to receive either a GMDP solution or a placebo; 63 patients were evaluated every third day for the mean daily number of episodes of nausea or vomiting, changes in clinical status, cell blood count and blood chemistry. A 95% inhibition of LDH release was noticed on A549 cells. Other cell lines were less sensitive to GMDP, with an observed 72% dose-dependent reduction in LDH activity.In vivo, LDH activity was decreased by 41% (+/−4%) (mean +/−SD) in all 21 subjects who were given 0.5–1.0 mg of GMDP daily. A lowering of LDH activity by 73.4% (+/−4%) was observed in 23 patients who received GMDP at a dosage of 1.5 mg/kg daily. Correspondingly, a 10% (+/−2%) increase in LDH activity was noticed in 19 patients who were given a placebo (P<0.01). During the follow-up period, the overall clinical condition of all patients treated with GMDP was improved. No side effects were observed. In nine patients who experienced nausea from tumor toxicity before treatment, the symptom subsided. In parallel, an extremely beneficial effect on lipids metabolism was noticed in all patients with elevated cholesterol and trigliceride levels. A dietary supplementation of GMDP has been shown to reduce systemic TNF-α cytotoxicity during tumor shock.     

New acellular pertussis vaccine, containing glucosaminylmuramyldipeptide

As shown in this work, the synthetic immunomodulator glucosaminylmuramyldipeptide (GMDP) can be included into acellular pertussis vaccine (APV). The optimal doses of GMDP, ranging from 0.001 to 0.0001 microg, have been found. These doses enhance the protective activity of APV, especially its low-active doses. GMDP decrease the manifestations of toxic, anaphylactogenic and pyrogenic properties of APV, which may lead to the decrease of the antigenic load of APV on the body of the vaccines and thus to lessening the side-effects of vaccination. GMDP has been shown to considerably increase, in comparison with common pertussis vaccine and APV, the percentage of phagocytizing leukocytes by day 14. The immunization of mice with APV with and without GMDP in doses of 0.01 and 0.001 microg leads to a change in T-lymphocyte/B-lymphocyte ratio in the population of spleen lymphocytes.     

Mechanism of action of N-acetylglucosaminyl-N-acetylmuramylalanyl-D-isoglutamine on the nonspecific anti-infection resistance of mice

The stimulating influence of glucose-containing muramyldipeptide (GMDP) on the nonspecific resistance of mice was shown to depend on the features of the pathogenesis of the infection. Thus, the intraperitoneal injection of GMDP increased the survival rate of mice infected with Escherichia coli, but had no stimulating effect on the resistance of the animals to Salmonella typhimurium natural infection in whose pathogenesis macrophages played an essential role. Experiments demonstrated that GMDP was capable of enhancing the ingestive function of macrophages, but did not increase their bactericidal activity with respect to this infection.     

Effects of Bordetella pertussis components on IgE and IgG1 responses

The effect of dermonecrotic toxin (DNT), fimbrial hemagglutinin (FHA), K-agglutinogen, lipopolysaccharide (LPS), and pertussigen from Bordetella pertussis on the production of IgE and IgG1 antibodies to hen egg albumin (Ea) was investigated in C57BL/6 mice. The IgE antibody contents were determined by passive cutaneous anaphylaxis (PCA) in the skin of Lewis rats, while the IgG1 antibody contents were determined by PCA reactions on the skin of mice using sera that had been heated for 3 hr at 56 C to destroy the IgE antibodies. Among the B. pertussis components tested, pertussigen was the most effective adjuvant for increasing the IgE and IgG1 antibodies to Ea. LPS also moderately increased both types of antibodies, and FHA slightly increased the IgG1 titers. When LPS was given 5 days before Ea, it suppressed both IgE and IgG1 titers while FHA had only slight adjuvant action on both type of antibodies. When each of the components was tested for its ability to modify the adjuvant action of pertussigen, it was found that only DNT interfered significantly with the adjuvanticity of pertussigen when given on the day of immunization with Ea. When the components were given 5 days before Ea, DNT produced significant suppression of only the IgG1 response. LPS, FHA, and K-agglutinogen did not significantly affect the adjuvant action of pertussigen.     

B subunit of Escherichia coli heat-labile enterotoxin as adjuvant of humoral immune response in recombinant BCG vaccination

The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. In this paper, the effect of LTB on the humoral immune response to recombinant BCG (rBCG) vaccination was evaluated. Isogenic mice were immunized with rBCG expressing the R1 repeat region of the P97 adhesin of Mycoplasma hyopneumoniae alone (rBCG/R1) or fused to LTB (rBCG/LTBR1). Anti-R1 systemic antibody levels (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA) were measured by ELISA using recombinant R1 as antigen. With the exception of IgM, LTB doubled the anti-R1 antibody levels in rBCG vaccination. The IgG1/IgG2a mean ratio showed that both rBCG/LTBR1 and rBCG/R1 induced a mixed Th1/Th2 immune response. Interestingly, anti-R1 serum IgA was induced only by rBCG/LTBR1. These results demonstrate that LTB has an adjuvant effect on the humoral immune response to recombinant antigens expressed in BCG.     

Role of group A streptococcal IgG Fc-receptor in induction of anti-IgG by immunization in the rabbit

Previous work has demonstrated that streptococcal IgG Fc-receptors (FcR) may trigger production of anti-IgG after immunization of rabbits with group A streptococci. This effect seemed dependent on in vitro binding of IgG, derived from the growth medium, to the vaccine strains. In the experiments presented here, IgG was eluted from streptococcal strains to be used for immunization of rabbits by 1 M KSCN and washing, a treatment which did not affect the capacity of the strains to bind newly added IgG. Using two IgG FcR-positive group A streptococcal strains (M-types 1 and 22) for intravenous immunization, anti-IgG was found in the sera of 26 out of 28 rabbits, examined 8 weeks after immunization. In contrast, anti-IgG was not induced in 16 rabbits receiving either group A, type T27 or group B, type Ia streptococci both of which lack surface FcR activity. Finally, immunization with purified streptococcal IgG FcR (0.35 mg, given subcutaneously combined with Freund's complete adjuvant and two weeks later intraconjunctivally without adjuvant) also induced anti-IgG. In all rabbits, anti-human rather than anti-rabbit IgG was detected. It is proposed that in vivo interaction between the bacterial FcR and rabbit IgG, resulting in conformation changes in IgG, is a prerequisite for the induction of anti-IgG. Thus, streptococcal triggering of anti-IgG, ascribable to IgG Fc-receptor activity and not requiring presence of foreign IgG, has been demonstrated in the rabbit.     

Role of group A streptococcal IgG Fc-receptor in induction of anti-IgG by immunization in the rabbit

Abstract Previous work has demonstrated that streptococcal IgG Fc-receptors (FcR) may trigger production of anti-IgG after immunization of rabbits with group A streptococci. This effect seemed dependent on in vitro binding of IgG, derived from the growth medium, to the vaccine strains. In the experiments presented here, IgG was eluted from streptococcal strains to be used for immunization of rabbits by 1 M KSCN and washing, a treatment which did not affect the capacity of the strains to bind newly added IgG. Using two IgG FcR-positive group A streptococcal strains (M-types 1 and 22) for intravenous immunization, anti-IgG was found in the sera of 26 out of 28 rabbits, examined 8 weeks after immunization. In contrast, anti-IgG was not induced in 16 rabbits receiving either group A, type T27 or group B, type Ia streptococci both of which lack surface FcR activity. Finally, immunization with purified streptococcal IgG FcR (0.35 mg, given subcutaneously combined with Freund's complete adjuvant and two weeks later intraconjunctivally without adjuvant) also induced anti-IgG. In all rabbits, anti-human rather than anti-rabbit IgG was detected. It is proposed that in vivo interaction between the bacterial FcR and rabbit IgG, resulting in conformation changes in IgG, is a prerequisite for the induction of anti-IgG. Thus, streptococcal triggering of anti-IgG, ascribable to IgG Fc-receptor activity and not requiring presence of foreign IgG, has been demonstrated in the rabbit.     

Endogenous Tumor necrosis factor alpha unmasks the binding sites for N-acetylglucosaminyl-(beta1-4)-N-acetylmuramyl-alanyl-D-isoglutamine on the surface of melanoma cells

The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 +/- 200 per cell) and affinity (Kd = 10 +/- +/- 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react in vitro with GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 +/- 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor alpha (TNF-alpha). The mechanism of the TNF-alpha action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.     

Immunological adjuvant effect of ginsenoside Rh4 from the roots of Panax notoginseng on specific antibody and cellular response to ovalbumin in mice

Ginsenoside Rh(4) (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD(50) value) being 407+/-12 microg/ml using a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice especially at a dose of 25 microg (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1 at a dose of 25 microg compared to the OVA control group (P<0.05 or P<0.01). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (Al(OH)(3) gel; P<0.01). These results suggest that 1 could be safely used as adjuvant with low or non-haemolytic effect.