Structural modification effects on bioactivities of the novel 15-mer peptide adjuvant.

Structure-function relationship studies for novel GMDP-peptide mimic, RVPPRYHAKISPMVN (RN-peptide) were performed on array of truncated and 'Ala-scan' analogues. The shortest peptide fragment possessing detectable affinity towards anti-GMDP-antibodies was demonstrated to be PRYH. RN-peptide analogues lacking up to 8 residues at C-terminus were found to retain adjuvant activity with the minimal active peptide being RVPPRYH. Evaluation of Ala-scan analogues highlighted that adjuvant activity is most critically dependent on both arginine residues, but also is sensitive to substitution of K9, I10, S11 and M13 amino acid residues, the functional importance of which was additionally confirmed by testing peptides truncated at both termini.

Refolding of scFv mini-antibodies using size-exclusion chromatography via arginine solution layer.

The method for refolding of mini-antibodies using size-exclusion chromatography via arginine solution layer was developed. This method allows to refold scFv, to separate both aggregated protein and low molecular weight compounds and to isolate functionally active protein preparation in monomeric form. The comparison of various scFv preparations isolated either from inclusion bodies or from soluble fraction revealed that refolded mini-antibodies demonstrate higher antigen-binding activity. Mini-antibodies refolded in the presence of arginine also demonstrate higher electrophoretic mobility during native PAGE in comparison with soluble cytoplasmic antibodies. Both soluble as well as refolded antibodies had similar CD spectra. Refolded mini-antibodies are storage-stable.

Glucosaminylmuramyl dipeptide potentiates the effects of tumor necrosis factor-alpha and cisplatin on transformed cells in vitro.

We studied the effect of combined treatment with cisplatin, glucosaminylmuramyl dipeptide, and TNF-alpha on viability of MCF-7, U-937, B16, and L-929 tumor cells, Ehrlich ascites carcinoma cells, and normal cells (human peripheral blood lymphocytes, peritoneal macrophages, and mouse bone marrow cells). Glucosaminylmuramyl dipeptide was nontoxic for normal and tumor cells, but promoted death of tumor cells after administration in combination with cisplatin and/or TNF-alpha. At the same time, glucosaminylmuramyl dipeptide did not modulate the cytotoxic effect of individual or combined treatment with cisplatin and TNF-alpha on normal cells. Administration of glucosaminylmuramyl dipeptide to cultured MCF-7 cells 20 h before the study increased the potentiating effect of muramyl peptide.

Mechanisms of defense formation against meningococcal infection in mice immunized with synthetic peptides.

Immunization of mice with synthetic peptide fragments of conservative sites of meningococcal outer membrane proteins led to defense formation against infection with virulent serogroup A and B Meningococci. The role of cellular immunity in the formation of defense against meningococcal infection after immunization with the peptides and the possibility of stimulating lymphocyte population with these peptides were demonstrated.

Protective properties of synthetic peptides of outer meningococcal membrane.

Peptide fragments of conservative sites of PorA, OpaB, and NspA proteins of the outer membrane of serogroup B meningococci were synthesized. These peptides caused a pronounced protective effect in immunized mice infected with virulent homologous and heterologous strains of serogroups B and A meningococci. The protective effect appreciably increased, if the studied peptides were associated in a polycomponent preparation, which can be used in the construction of meningococcal bivalent B+A vaccine.

Quantitative immunoassay of biotoxins on hydrogel-based protein microchips.

Three-dimensional gel-based microchips with immobilized proteins were used for quantitative immunoassay of a series of plant (ricin and viscumin) and bacterial (staphylococcal enterotoxin B, tetanus and diphtheria toxins, and lethal factor of anthrax) toxins. It was shown that different types of immunoassays (direct, competitive, and sandwich type) could be carried out on gel microchips. As shown by confocal microscope studies, antigen-antibody interactions involving the formation of tertiary antibody-antigen-antibody complex occur in the whole volume of microchip gel elements. Sandwich assay on microchips with immobilized antibodies provided the highest sensitivity of detection (0.1 ng/ml for ricin). Antibodies labeled with fluorescent dyes, horseradish peroxidase conjugates, or biotinylated antibodies with subsequent treatment with labeled avidin were used as developing antibodies. The results of immunoassays were recorded using fluorescence, chemiluminescence, or matrix-assisted laser desorption ionization mass spectrometry directly from microchip gel elements. Gel microchips with immobilized capture antibodies were used to analyze the sample simultaneously for the presence of all six biotoxins with the same sensitivity as that for any single toxin.

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide.

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

Destabilase from the medicinal leech is a representative of a novel family of lysozymes.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

Specific binding of glucosaminylmuramyl peptides to histones.

Intracellular N-acetylglucosaminylmuramyl peptide-binding proteins of murine macrophages and myelomonocytic WEHI-3 cells were characterized. SDS-PAGE and Western blotting revealed proteins with molecular masses of 18, 32 and 34 kDa retaining the ability to specifically bind glucosaminylmuramyl dipeptide. The inhibition analysis demonstrated that only biologically active muramyl peptides but not inactive analogs or fragments of glucosaminylmuramyl dipeptide could inhibit glucosaminylmuramyl dipeptide-binding to these proteins. Purification of these proteins and sequencing of peptides obtained after in-gel trypsin digestion enabled us to identify the above mentioned proteins as histones H1 and H3. These findings suggest that nuclear histones might be target molecules for muramyl peptides.

Endogenous tumour necrosis factor-alpha sensitise melanoma cells to glucosaminylmuramyl dipeptide.

Flow cytometry was used to demonstrate that cultured human melanoma BRO cells expressed membrane-bound tumour necrosis factor-alpha (TNF-alpha) and were able to release TNF-alpha upon treatment with glucosaminylmuramyl dipeptide (GMDP). The released TNF-alpha was shown to prime melanoma cells, previously unable to respond to GMDP by increasing expression of melanoma-associated antigens, making them sensitive to GMDP treatment.

Glucosaminylmuramyldipeptide-induced changes in phenotype of melanoma cells result in their increased lysis by peripheral blood cells.

Flow cytometry was used to show that biologically active N-acetylglucosamine-containing muramylpeptides (GMPs) induced in vitro dose-dependent increase in the expression of tumor-associated antigens (TAAs) characteristic for colon and mammary gland carcinomas, melanoma and lung adenocarcinoma. Forty to two hundred percent enhancement in TAA-expressing cells was observed after 18-48 h incubation with GMPs. In contrast, MHC class I antigen expression was not altered. Using MTT and chromium-release assays, melanoma cells treated in vitro with GMDP were shown to be more susceptible to killing by peripheral blood cells of healthy donors than non-treated cells. Fractionation of blood cells revealed that platelets were responsible for this effect.

N-acetylglucosamine-containing muramyl peptides directly affect macrophages.

In this study flow cytometry was used to show that macrophages were the major population of murine peritoneal exudate cells (MPEC), increasing Ia expression upon treatment with N-acetylglucosaminyl-beta 1-4-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Modulation of Ia expression resulted from direct action of GMDP on macrophages, rather than from effect of cytokines released by T-cells. The effect of GMDP on two populations of macrophages, namely, slow and rapid responding, was studied in detail. Rapid responding cells were represented by Ia-positive macrophages: GMDP augmented their Ia expression. In contrast, slow responding subpopulation was represented by initially Ia-negative macrophages, in which GMDP induced de novo synthesis of Ia-antigens. The ability to induce Ia expression was also characteristic for other adjuvant-active N-acetylglucosamine-containing muramyl peptides (GMPs). Macrophages were shown to engulf GMPs by endocytosis. Activation of macrophages by GMDP resulted in an increase in their phagocytic activity.

Production and characterization of monoclonal anti-idiotypic antibodies to muramylpeptide.

Hybridomas producing monoclonal anti-idiotypic antibodies (anti-id MAbs) to N-acetylglucosaminyl-beta 1-4-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP) were developed. Three clones of hybridomas demonstrated the properties characteristic for the Ab2 beta type of anti-id antibody: they bound to Fab-fragments of high-affinity MAb to GMDP; dose-dependent inhibition of this binding by GMDP was observed; immunization of mice with these MAbs resulted in production of GMDP-specific antibodies. When these antibodies were used to stain blots from SDS-PAGE of macrophage lysate, the same receptor proteins were specifically stained as upon staining with 125I-labelled GMDP derivative.

Biochemical characterization of glucosaminylmuramyldipeptide binding sites of murine macrophages.

By using radioligand analysis, murine peritoneal macrophages were shown to express several hundred high-affinity cell surface GMDP-binding sites (Ka 350 pM). Photoaffinity labeling followed by SDS-PAGE enabled us to identify 32-34 and 38 kDa proteins inside these cells that bound GMDP specifically.

Immunoregulatory properties of hexapeptide isolated from porcine bone marrow cell culture.

Myelopeptide 1 (MP-1) is hexapeptide originally isolated from porcine bone marrow cell culture. It was synthesized and its immunoregulatory properties were studied. MP-1 caused a 1.5-2-fold dose-dependent increase of antibody production in the culture of mouse immune lymph node cells. It abolished Con A induction of T suppressors in the suspension of mouse spleen cells and counteracted the inhibitory effect of T suppressors on antibody production. The inoculation of MP-1 (1 x 10(-9) g/mouse) to mice two weeks after their gamma-irradiation (2 Gy) resulted in an increase of antibody production up to 80.2 +/- 15.5% as compared to that in the irradiated control 37.6 +/- 12.0%. Immunofluorescent analysis revealed the specific binding of MP-1 with receptors on the target cells in the suspension of mouse spleen cells. It is supposed that MP-1 participates in the immunoregulatory processes in the living organism.

Albumin-like glycoprotein from human fetal tissue.

Albumin-like glycoprotein (Gp66) with a molecular mass of 66 kDa has been isolated from human fetal tissue by size-exclusion, ion-exchange chromatography and reverse-phase HPLC. Reactivity of Gp66 with antiserum raised against the major protein components fraction of human fetal serum was observed. The N-terminal 35 amino acid residues of Gp66 were identical to human serum albumin. Meanwhile Gp66 differed from albumin by a/ the presence of 3-5 Trp residues instead of 1 according to fluorescence and UV-spectra, b/ the glycosylation pattern: bi-, tri-, and tetraantennary sialooligosaccharides of a complex type were present. Isoelectric focusing revealed 4 isoforms (pI ranging within 4.8 to 5.1) of Gp66. Gp66 (but not asialo-Gp66) was able to inhibit the cytotoxic effect of TNF against the tumor cell line L929. Inhibition of WEHI-3 and L929 tumor cells proliferation by Gp66 was similar to that of albumin.

Bispecific monoclonal antibodies to human interleukin 2 and horseradish peroxidase.

Hybrid hybridomas (tetradomas), producing bispecific monoclonal antibodies (bmabs) binding to both interleukin 2 and horseradish peroxidase were obtained by fusing IL 2-specific and HRP-specific hybridomas. Parental hybridomas were labelled prior to the fusion with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate. Bifluorescent (tetradoma) cells were sorted out using fluorescence activated cell sorter. Two clones, designated D8C1/G and H7C11/H were shown to secrete bmabs over at least 6 months growth in culture. Bmabs have been purified from mouse ascites by ammonium sulfate precipitation and affinity chromatography on immobilized peptide, modelling corresponding IL 2 epitope. The purity of antibodies obtained was characterized by capillary electrophoresis. The possibility of using these antibody preparations in rIL 2 analysis was evaluated in two types of EIA: direct and competitive solid-phase EIA. Both assays had similar sensitivity of about 1.5-4 ng IL 2 per ml.

Muramyl peptide-binding sites are located inside target cells.

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide-binding sites were shown to be located inside T-lymphocytes, macrophages and neuroblastoma cells, but not inside B-cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEHI-3 cells with Kd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D-configuration of isoglutamine residue are important.

Muramylpeptides augment expression of Ia-antigens on mouse macrophages.

The muramylpeptide N-acetylglucosaminyl-beta 1----4-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) was shown by flow cytometry to produce a dose-dependent increase in the expression of Ia-antigens by mouse peritoneal macrophages in vitro. Muramyldipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) had a similar, but weaker, effect. GMDP also induced expression of Ia-antigens by murine peritoneal macrophages in vivo. GMDP acted directly on the macrophages because Ia-antigen expression by cells of the cloned mouse myelomonocytic line WEHI-3 was also induced. Expression of the interleukin-2 receptor on the surface of the macrophages was also stimulated by GMDP, indicating that GMDP may influence development of the immune response through this mechanism.