[Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein].

INTRODUCTION: A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants. The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid. MATERIALS AND METHODS: An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated. RESULTS: Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60. CONCLUSIONS: Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

Wound healing activity of aqueous dispersion of fullerene C60 produced by "green technology".

In addition to exhibited antioxidant and anti-inflammatory activity, fullerene C60 is a promising wound healing agent. An important stage in the production of fullerene-based ointments is the stability of the aqueous fullerene dispersion (AFD) with minimum size of colloidal fullerene aggregates and sufficiently high concentration. To achieve these parameters tangential flow filtration of fullerene C60 was used ("green technology"). As estimated by small-angle neutron scattering and dynamic light scattering purified AFDs with narrow-size distribution nanoclusters have a size of 6 nm and are assembled into agglomerates which reach a size of 150 nm. The ability of the AFD to exhibit regenerative activity was studied using the animal wound model. This study shows for the first time that the fullerene-based composition stimulates the healing of wounds of various origins. We assume that the mechanism of the AFD wound-healing activity is associated with the aryl hydrocarbon receptor and macrophages activity.

[Prospects For the Use of Peptides against Respiratory Syncytial Virus].

The human respiratory syncytial virus (RSV) is one of the most common viral pathogens that affects the lower respiratory tract and could be a reason of bronchiolitis and/or pneumonia. Currently, there are no available effective ways of treating the RSV infection. Attempts to develop preventive vaccine have been unsuccessful. The only therapeutic agent used for RSV treatment is virazole (ribavirin); however, it induces adverse effects. Medications based on neutralizing monoclonal antibodies, such as IGIV (Respigam), palivizumab (Synagis), and MEDI-524 (Numab), are under clinical trials; however, their use will be limited by their high cost. One of the promising approaches for antiviral therapy is the use of natural peptides (defensins and cathelicidins), or their synthetic analogs. The majority of currently described antiviral peptides are developed against the human immunodeficiency virus, the herpes simplex virus, and the influenza virus. At the same time, a body of experimental data evidencing anti-RSV activity of peptides has been accumulated. The main advantages of peptide drugs are their wide spectrum of antiviral activity and low toxicity. However, there are obstacles in implementing peptide-based drugs in clinical practice. Due to their low resistance to the action of serum proteases, most authors consider peptides promising only for local application. Given that RSV affects the epithelium of the respiratory tract, where the protease activity is lower than in the systemic circulation, it is possible to develop locally active peptide drugs, for example, as inhalation forms. Their stability could also be increased by the synthesis of dendrimer peptides and by the development of recombinant peptides as precursor proteins. Anti-RSV peptides can be divided into several groups: (1) attachment and/or fusion blockers; (2) peptides displaying direct virucidal activity, disrupting the viral envelope. Such peptides, which suppress early stages of the viral life cycle, are considered prophylactic agents. However, for several peptides, their immunoregulatory properties have been described, which opens the possibility for therapeutic use. This review summarizes the information on the antiviral properties of such peptides and mechanisms of their action and describes the prospects of the future development of antiviral peptides.

Synthesis and evaluation of novel lipopeptide as a vehicle for efficient gene delivery and gene silencing.

Nucleic acid-based therapeutics have recently emerged as a new class of next generation agents for treatment and prevention of viral infection, cancer, and genetic disorders, but their wide use is limited by their relatively weak delivery into target cells. Usage of synthetic cationic amphiphiles with peptide hydrophilic domain as agents for non-viral gene delivery is an attractive approach. We developed the schemes for the synthesis of aliphatic peptides with different length of the hydrocarbon chains in hydrophobic domains and different amino acids in polar head. For the obtained derivatives we determined transfection efficiency, critical vesicle concentration, particle size, ζ-potential and aggregates stability. We have found that the transfection efficiency is increased if the ornithine is a part of polar head in an amphiphile. The most promising amphiphile for liposomal formation OrnOrnGlu(C16H33)2 was examined more carefully. It has been shown that the lipopeptide possesses low toxicity (in vitro and in vivo) and high transfection efficiency with pDNA and siRNA in different cell lines. In addition, the production of liposomes based on this lipopeptide is simple, quick and cheap. Thus OrnOrnGlu(C16H33)2 is a promising vehicle for gene delivery and gene silencing.

[Anti-peptide antibodies specifically recognize the L1 protein of human papilloma virus type 31].

Antibodies that specifically recognize the capsid protein (L1) of human papillomavirus (HPV) are an important tool necessary for designing vaccines against HPV infection. In this work, we have predicted and synthesized peptide fragments mimicking B cell epitopes of L1 HPV type 31 (sequences 49-65, 131-145, 172-189, 349-362 and 402-414), and conjugated their to KLH and BSA to generate the L1-31-specific anti-peptide antibodies in mice. Variants of recombinant L1-31, including full-size and mutants with C-terminal single amino acid changes and deletions and full-size L1-16 were produced in the yeast using monitoring with L1 HPV16-specific monoclonal antibody. Testing of anti-peptide antisera in ELISA showed that antibodies to peptides 49-65 and 172-189 were capable to recognize specifically L1-31 protein, but not L1-16 one. Such antibodies may be used for assay of L1-31 production in various expression systems.

[Chemical modification of allergen leading to changes in its epitopic activity]. / Khimicheskaia modifikatsiia allergena, vedushchaia k izmeneniiu ego épitopnoi aktivnosti.

Modification of a model allergen ovalbumin (OA) with succinylation led to a decrease of its allergenicity measured by passive cutaneous anaphylaxis reaction, RAST inhibition assay and basophil histamine release. Modified OA stimulated OA-specific T-cell hybrid 3DO-548 to produce IL-2 at the same level as in case of non-modified OA. Modified OA did not induce anti-OA IgE, but did induce anti-OA IgG antibodies. This approach to chemical modification of allergen-selective blockade of B-cell epitopes while not affecting T-cell epitopes suggests new opportunities in creation of safe and effective allergovaccines.

Modulation of Basophil Histamine Release and T Cell Reactivity by Chemically Modified Allergen.

Modification of a model allergen ovalbumin (OA) with succinylation (Suc-OA) led to inhibition of allergen histamine releasing activity as it was tested on basophils of OA-sensitive patients. A whole blood leukocyte histamine release was performed by glass fibre based histamine assay. IgE-binding activity of Suc-OA was significantly reduced as it was shown by RAST inhibition technique. Suc-OA stimulated OA-specific T cell hybrid 3DO-548 and ACP:LK35 to produce cytokine release at the same level as in the case of non-modified OA. Succinylation of OA was concluded to result in selective blockade of B cell and preservation of T cell epitopes of the allergen suggesting a new approach for allergen specific immunotherapy.

Targeting of liposomes to HIV-1-infected cells by peptides derived from the CD4 receptor.

Liposomes can be targeted to HIV-infected cells by either reconstituting transmembrane CD4 in the membrane or covalently coupling soluble CD4 to modified lipids. We investigated whether synthetic peptides could be used as ligands for targeting liposomes. A synthetic peptide from the complementarity determining region 2 (CDR-2)-like domain of CD4 could bind specifically to HIV-infected cells and mediate the binding of peptide-coupled liposomes to these cells. A peptide from the CDR-3-like domain of CD4 inhibited HIV-induced syncytia formation, but failed to target liposomes to infected cells. This apparent discrepancy may be due to the requirement for a conformational change in the CD4 receptor for the CDR-3 region to interact with the HIV envelope protein. Our results demonstrate the feasibility of using synthetic peptides to target liposomes containing antiviral drugs to HIV-infected cells.

[Fusion of negatively-charged liposomes under the effect of peptides from the N-terminal fragment of the HIV-1 transmembrane protein]. / Sliianie otritsatel'no zariazhennykh liposom pod deistviem peptidov iz N-kontsevogo fragmenta transmembrannogo belka VICh-1.

The effect of a series of synthetic peptides mimicking the N-terminus of HIV transmembrane glycoprotein (gp41) on fusion of negatively charged liposomes consisting of phosphatidylcholine, phosphatidylethanolamine and cardiolipin at a 2:3:5 molar ratio, respectively, has been studied. Peptides P514 and P385 (residue 517-538), lysine and arginine at the C-terminus, respectively, with the amino acid sequence completely corresponding to the N-terminus of gp41 displayed the highest fusogenic activity. The extent of fusion was significantly increased at mild acidic pH (6.0). Acidification particularly influenced the fusogenic activity of P514. Modification of the N- and C-termini of fusion-active peptides by proteins and synthetic polymers blocked the fusion activity. The fusogenic properties of peptides depended on the chain length: P411 consisting of nine hydrophobic amino acid residues had no fusogenic activity, while P415, an 11-member peptide, effectively fused liposomes. The fluorescent probe ANS was used to monitor the hydrophobicity of these peptides. The hydrophobicity of P514 increased appreciably with a change in pH from 6.0 to 7.5. Peptides P514 and P385 induced the leakage of the aqueous contents from liposomes at neutral pH and caused a small, but detectable leakage at acidic pH. Structural and molecular factors influencing the peptide-induced liposome fusion are discussed.

[Identification of immunoreactive epitopes in proteins coded by gag, env, and pol genes of the type I human T-lymphotropic virus (HTLV-I) using synthetic peptides]. / Identifikatsiia immunoreaktivnykh épitopov v belkakah, kodiruemykh genami gag, env i pol T-limfotropnogo virusa cheloveka tipa I (HTLV-I) s ispol'zovaniem sinteticheskikh peptidov.

Reactivity of 26 synthetic peptides that comprise 12 to 26 amino acid residues corresponding to segments of the gag p19, env gp46, and pol proteins of human T-lymphotropic virus type I toward 31 positive sera was studied using enzyme-linked immunosorbent assay. Specific reactivity with high titers of antibodies (presented in reciprocal dilution values) was detected for the synthetic peptides corresponding to fragments 110-130 and 100-130 (titers up to 4050) of p19, 174-197 (up to 800), 186-201 (up to 4050), 191-215 (up to 1350), 242-257 (up to 800), and 272-292 (up to 450) of gp46. Immunoreactivity of seven peptides, fragments of pol-proteins, was weak. New linear epitopes in the regions 145-158, 272-277, and 292-300 of gp46 were detected. In addition, location of the known linear epitopes in p19 and gp46 was refined on the basis of comparative study of overlapping peptides from these proteins.

Immunological properties of allergen chemically modified with synthetic copolymer of N-vinylpyrrolidone and maleic anhydride.

Several conjugates of model allergen ovalbumin (OA) and the copolymer of N-vinyl pyrrolidone and maleic anhydride (VMA) modified with epsilon-aminocaproic acid (Acp) were prepared in different OA/Acp-VMA ratios. All conjugates were separated by ultrafiltration and analyzed by HPLC. Their compositions were determined by amino acid analysis and UV spectrometry. To detect immunogenicity, all conjugates were injected intraperitoneally into (CBAxC57BL/6)F1 mice three times in 3-week intervals in OA doses equivalent to 0.5, 10, and 100 micrograms/mouse. Only the conjugate containing 20%OA (OA(20%)-Acp-VMA) did not induce significant quantities of anti-OA IgE, but did induce anti-OA IgG antibodies in dose-dependent manner comparable to that of unmodified OA. Mixtures of OA and Acp-VMA or OA modified only with VMA without Acp activation with Acp induced dose-dependent anti-OA IgE and IgG antibody formation comparable to that of OA. Using passive cutaneous anaphylaxis, RAST inhibition and leukocyte histamine release, a significant reduction of allergenicity was noted using OA(20%)-Acp-VMA. This conjugate stimulated activation of the OA-specific T-cell hybrid 3DO-548 comparable to that of unconjugated OA. During experimental allergen-specific hyposensitization with OA(20%)-Acp-VMA, suppression of anti-OA IgE response and elevation of anti-OA IgG responses were noted when compared with unmodified OA. Selective blockade of B-cell epitopes of allergen may occur using the carrier Acp-VMA to reduce allergenicity while not affecting T-cell epitopes, thereby preserving immunogenicity. This approach of chemical modification of allergen suggests new opportunities in the creation of preparations for allergen-specific immunotherapy.

[Cross reactivity and amino acid structural homology of immunodominant peptide fragments of structural viruses from the human retroviruses HTLV-I, HTLV-II, HIV-1, and HIV-2]. / Perekrestnaia reaktivnost' i gomologiia aminokislotnoi struktury immunodominantnykh peptidnykh fragmentov strukturnykh belkov retrovirusov cheloveka HTLV-1, HTLV-II, HIV-1 i HIV-2.

Twelve synthetic peptides corresponding to 9 immunodominant regions of structural proteins of human retroviruses HTLV-I, HIV-1, and HIV-2 were studied in enzyme-linked immunosorbent assay for cross reactivity with heterotypical for each peptide anti-retroviral antibodies. The search of amino acid homologies was carried using the special computer program followed by the correspondence analysis of the discovered homologies and immunodominant fragments. It was found that peptides 100-130 p19 gag HTLV-I, 376-392 gp21 env HTLV-I, 381-400 gp21 env HTLV-I, 306-328 gp120 env HIV-1, 495-516 gp120 env HIV-1, 584-612 gp41 env HIV-1, and 581-603 gp36 env HIV-2 have type-specific reactivity and also cross react with 3-54% human sera containing antibodies against heterotypical retroviruses. On the other hand, peptides 120-130 p19 gag HTLV-I, 176-201 gp46 env HTLV-I, 291-312 gp46 env HTLV-I, 330-363 p24 gag HIV-1, and 602-624 gp41 env HIV-1 have shown no cross reactive properties; they may be effectively used for type-specific and differential serodiagnosis of human retroviral infections.

[Immunoreactivity of synthetic peptides, corresponding to B-cellular epitopes of human type I T-lymphotrophic virus structural proteins]. / Immunoreaktivnost' sinteticheskikh peptidov, sootvetstvuiushchikh B-kletochnym épitopam strukturnykh belkov T-limfotropnogo virusa cheloveka I tipa.

The immunoreactivity of 25 synthetic peptides corresponding to amino acid fragments of the HTLV-I structural proteins p19 gag, gp46 and gp21 env were studied in enzyme linked immunosorbent assay using a serum panel of 70 reference positive specimens with anti-HTLV-I antibodies. The location of the synthetic peptides containing the B-cell epitopes of HTLV-I was established. Anti-HTLV-I antibodies effectively recognized these peptides. The significance of some amino acids for forming the HTLV-I antigenic determinants was estimated. The synthetic peptides with amino acid sequences 100-130 p19 gag and 176-201 gp46 env were found to have most immunoreactivity (90-99% recognition by sera of HTLV-I infected patients) and mimic the immunodominant B-cell epitopes of HTLV-I structural proteins.

[A synthetic peptide-based immunoenzyme test system for the joint detection of human retrovirus (HTLV-1 and HIV-1) antibodies]. / Immunofermentnaia test-sistema dlia sochetannogo vyiavleniia antitel k retrovirusam cheloveka (HTLV-1 i HIV-1) na osnove sinteticheskikh peptidov.

The study was made to develop an immunodiagnostic test system based on synthetic peptides able to detect in the same assay the total concentration of heterospecific antibodies against human retroviruses HTLV-1 and HIV-1. Three panels of reference-sera contained antibodies to HTLV-1 (70 specimens), HIV-1 (50 and 16 specimens) and 4 synthetic peptides corresponding to protein fragments p19 gag and gp46 env HTLV-1, gp120 and gp41 env HIV-1. Immune reactivity of the peptides with reference sera was measured in the immunoassay. It is established that relevant peptides mimic immunodominant B-epitopes of structural proteins HTLV-1 and HIV-1 and are recognized specifically by relevant antiviral antibodies. The enzyme immunoassay test system has been designed using a peptide combination in a single antigen complex. The system showed high diagnostic sensitivity.

Inhibition of human immunodeficiency virus type 1 (HIV-1) penetration into target cells by synthetic peptides mimicking the N-terminus of the HIV-1 transmembrane glycoprotein.

To investigate the mechanism of action of the 22-amino-acid HIV fusion peptide on HIV infection, we studied its influence on virus adsorption and HIV-induced syncytium formation. The effect of the peptide preparations on the synthesis of viral antigens in HIV-infected cell cultures was determined by antigen capture assay, and the inhibition of proviral DNA synthesis was detected by hybridization with a HIV-specific oligonucleotide probe after PCR amplification. Fusion peptides inhibited HIV-induced syncytium formation and antigen production in lytic infected cells, and this effect was increased in conjugation with bovine serum albumin or with synthetic net-charged polymer by its C-terminus. The association of peptide with carrier by N-terminus, or with positive-charged polymer or gelatin completely abolished its effect on HIV infection. No peptide preparations influenced HIV-1 chronically infected cells. Because peptide preparations blocked the HIV-specific DNA synthesis 2 hr after infection without influencing virus adsorption and reverse transcription, we concluded that the block of infection occurred during the penetration of virions through the cell membrane. On the basis of results obtained we propose that our peptide preparations could be used for anti-HIV chemotherapy. The possibility of the existence of receptors for gp41 N-terminal region on target cell membrane is discussed.