[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.

[Influence of membrane-active polyanions on various stages of the human cytomegalovirus life cycle in vitro].

Human cytomegalovirus (CMV), an agent of infection (CMVI), lethally dangerous for immune deficient neonates and adults was investigated in vitro as a target for a therapeutic effect of new membrane-active polyanionic compounds (MPC). Previous studies on the alicycle- and sulfate-modified carboxy-MPCs revealed a well-defined tendency of the anti-CMV activity amplification in parallel with increasing of the content of sulfate groups, enhancing the negative charge of the macromolecule. The dominating role of the electrostatic factor was confirmed by the highest activity of AS-688, compound with maximum sulfation among the tested MPCs. Its selectivity index (SI) of the CMVI inhibition in human diploid fibroblast cells reached 5450, 7500, 250 and 4286 in the microbicidal, viricidal, prophylactic and therapeutic schemes of the experiment respectively. The antiviral activity at the first, second and third schemes was explained by the polyanion-typical potential of electrostatic neutralization of the countercharged virions and prevention of the virus adsorption on the cell membranes (in competition with heparin sulfate, a cellular receptor of CMV), whereas the therapeutic effect required the ability of MPC to influence the intracellular stages of the CMV life cycle. The PCR and immunochemical assays revealed an inhibitory action of AS-688 on replication of the viral DNA and the following synthesis of the late viral protein gB with efficiency similar to that of gancyclovir (GCV). However, in contrast to GCV, acting as inhibitor of enzyme (viral RNA-polymerase) factor of the biosynthesis, the therapeutic activity of MPC could be interpreted by competition with viral RNA/DNA due to the specific character of the MPC molecular basis, initially constructed on the principle of nucleic acids backbone and charge adjustable imitation. This mechanism assuming reduction of the cytotoxicity risks, explained the experimentally observed fact of low cytotoxicity of MPCs and possible achievement of high SI. The MPC ability to penetrate into the cells without disruption of cellular membrane permeability was confirmed in experiments with the fluorescent-labeled derivate AS-679, structurally and functionally related to AS-688. In the light of the previously described HIV inhibiting properties of AS-688, AS-679 and MPC analogous, the results could be considered prospective in development of new highly effective agents for combined antiviral protection.

[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.