Hepatitis C is one of the most common social diseases in the world. The improvements in both the early diagnostics of the hepatitis C and the treatment of acute viremia caused by hepatitis C virus are undoubtedly an urgent task. In present work, we offered the micro- and nanotraps for the capturing of HCV. As a capturing moiety, we designed and synthesized in E. coli a fusion protein consisting of large extracellular loop of CD81 receptor and streptavidin as spacing part. The obtained protein has been immobilized on the surface of PLA-based micro- and nanoparticles. The developed trapping systems were characterized in terms of their physico-chemical properties. In order to illustrate the ability of developed micro- and nanotraps to bind HCV, E2 core protein of HCV was synthesized as a fusion protein with GFP. Interaction of E2 protein and hepatitis C virus-mimicking particles with the developed trapping systems were testified by several methods.

We investigated immunogenic properties of proteins bound with nanoparticles. A process for producing spherical nanoparticles having size of 20 microns by polymerization of lactic acid and an optimal method of nanoparticle surface activation were described. Activated nanoparticles were used for covalent binding of model fusion protein comprising sequences of human beta-2 microglobulin and green fluorescent protein. It is shown that the nanoparticles were able to bind 3 micrograms of the protein per 1 mg of the polymer. According to the results of confocal microscopy and electrophoresis the protein is firmly adsorbed on the surface of the granules. F1 (CBA x C57BL) mice were subjected to intraperitoneal immunization with fusion protein modified nanoparticles and equivalent mixture of unmodified nanoparticles and unbound fusion protein. Blood was taken at 2 weeks after three-time intraperitoneal immunization. Antibody level to model protein was determined in mouse sera using enzyme-linked immunosorbent assay. Each of experimental and control groups comprised 39 animals. The validity of the results was evaluated using the Mann–Whitney test. It is shown that the average antibody level in the control group was 1.8 times greater than that in the experimental group. The diffe rence was significant (p < 0.004). We discuss the significance of the results in terms of development traps capable to bind virus particles in blood and to provide immune response.

The current study was a part of the project on generating viral particle traps occurring due to covalent immobilization on the interface of recombinant virus-specific polymer-based nano- and microparticles. It is assumed that protein-particle conjugates could be able to bind virions followed by engulfment by immune cells. The study was aimed to examine the effect of polylactic acid (PLA) and PLA block-copolymer with polyethylene glycol (PLA-PEG)-based micro- and nanoparticles on the cellular immune response against polymeric particle-bound antigen. Materials and methods. A recombinant chimeric protein beta-2-microglobulin — green fluorescent protein (β2M-sfGFP) was obtained by affine chromatography. The recombinant protein was immobilized onto the polymer particles, which were further used for mice immunization. Female F1 hybrid mice (CBA x C57BL) in experimental and control groups consisted of 4–6-month-old 15 animals (weighted 20–25 g). Intracellular cytokine staining was used to evaluate the cellular immune response. Results and discussion. It was shown that the nanoparticles of PLA block-copolymer with polyethylene glycol (PLA-PEG) were able to bind 10 microgram protein per 1 mg polymer. The polylactic acid nanoparticles were able to bind 2,3 microgram protein per 1 mg polymer. In experiment, mice in group 1 were immunized with 100 nm PLA-PEG particle-β2M-sfGFP conjugate, in group 2 — with same particles together with soluble β2M-sfGFP. In group 3, mice were immunized with 1400 nm PLA particles-β2M-sfGFP conjugate, and in group 4 — with same particles together with soluble protein. The spleens isolated 2 weeks after the four-time intraperitoneal immunization. Comparison of immune response between groups was assessed by nonparametric Kruskal–Wallis criterion with Tukey correction. It was shown that the number of antigen-specific CD4+ T cells produced to model protein was significantly higher after immunization with particle-β2M-sfGFP conjugate, as compared to control groups, wherein immunization was performed with a mixture of protein and unmodified particles (p 0.001). It was found that the number of antigen-specific CD8+ T cells formed against β2m-sfGFP did not differ between all groups examined.

The study is aimed at evaluating the influence of composition and size of micro- and nanoparticles (MPs and NPs) on the immunogenicity of protein antigen associated with them. For comparative analysis, MPs and NPs based on poly(lactic acid) (PLA) and block copolymer of poly(ethylene glycol) with poly(lactic acid) (PEG-b-PLA) were obtained. Recombinant human beta2-microglobulin fusion protein with superfolder green fluorescent protein (β2M-sfGFP) was used for covalent modification of all types of polymer particles. Immobilization of the model protein β2M-sfGFP was carried out on the surface of the particles through the reaction of activated esters with the amino groups of the protein. Immunization of mice using complex antigen (β2M-sfGFP protein immobilized on the surface of MPs and NPs of different compositions) was carried out in four steps. Immunogenicity was assessed by the level of specific antibodies to sfGFP using enzyme-linked immunoassay. The results showed a significant increase in antibody levels in the control groups, which were immunized with a mixture of model protein and particles of different nature and sizes, compared to the experimental groups, which were immunized with conjugates of the corresponding particles with model protein. In the experimental groups, the highest number of specific antibodies was detected in the case of immunization of mice with the conjugate of protein and PLA or PEG-b-PLA-based NPs. The introduction of PEG block into the PLA composition did not significantly affect the immunogenicity of the protein, while the particle size was of significant importance. Protein bound to PLA- or PEG-b-PLA-based NPs showed higher immunogenicity compared protein bound to MPs of the same compositions, which can be used for practical purposes to develop vaccines (NP-protein) or “trapping systems” (MP-protein) that bind infiltrating viruses.

The effect of the composition and size of polymeric particles on the immunogenicity of the fibrillogenic β2-microglobulin protein immobilized on their surface was studied. For this purpose, nanoparticles (NP) based on a copolymer of L-glutamic acid and L-phenylalanine (P(Glu-co-Phe)) and a block copolymer of poly(ethylene glycol) with poly(lactic acid) (PEG-b-PLA) as well as microparticles (MP) based on poly(lactic acid) (PLA) were selected. Materials and Methods. α-L-amino acid copolymer-based nanoparticles were prepared by gradient phase inversion, and PEG-b-PLA-based nanoparticles by nanoprecipitation. Double emulsion method was used to form polymeric microparticles based on PLA. Recombinant chimeric model protein beta-2-microglobulin-green fluorescent protein (β2M-sfGFP) was used to covalently modify all types of polymeric particles followed by immunization of four groups of laboratory animals equal in number. An enzyme immunoassay method was used to evaluate the humoral immune response. Results and discussion. In three experimental groups, mice were immunized using poly(amino acid)-based (NP-PAA) and PEG-b-PLA (NP-PLA) nanoparticles as well as PLA microparticles containing immobilized β2M-sfGFP on the surface. The control group was immunized using a physical mixture of PEG-b-PLA nanoparticles and free β2M-sfGFP. The highest level of antibodies to sfGFP in blood serum was found when mice were immunized with a mixture of protein and nanoparticles. When mice were immunized with β2M-sfGFP-modified nanoparticles, the amount of antibodies to sfGFP was statistically significantly lower (p < 0.001) compared to the control group. However, the groups immunized with nanoparticles of similar size but different composition conjugated to the model proteins did not differ significantly among themselves. It was also found that the size of the particles affects the immunogenicity of the associated protein. A similar pattern of relative antibody content in the sera of mice was maintained at all steps of immunization.