Novel polypeptide-comprising biopolymer systems.

Covalent bioconjugation between anionic polyelecrolytes and polypeptide antigens chemically synthesized by solid-phase chemistry, were studied in hydrated reversed micelle systems. The epitops of Foot-and-Mouse disease virus VP1 protein (40--60 and 135--160 residues) were used as polypeptide antigens. The polypeptide-comprising Biopolymer Systems were obtained by two methods: 1) Inclusion of peptides into electrostatic polyelectrolyte complexes of polycations with proteins, 2) Inclusion of peptides into Cu(+2)-induced polyelectrolyte complexes of polyanions with similarly (anionic) charged proteins. The immunogenetic properties of polymer-peptide conjugates and peptide-comprising Biopolymer Systems were investigated and the specificity of antibodies produced was analyzed. Polymer-Peptide conjugates, as well as peptide-comprising Biopolymer Systems appeared to possess a high peptide specific immunogenecity even without the addition of traditional adjuvants. It was found that PE-BSA.FMD.Ag conjugates conferred effective immunoprotection against Foot-and-Mouth disease virus.

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and bovine serum albumin.

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and negatively charged bovine serum albumin (BSA) was studied in neutral water in the presence of Cu2+. Depending on the concentration of Cu2+, the reaction between PAA-Cu2+ complexes and BSA appeared to follow one of two possible paths. At low Cu2+ concentrations (nCu/nAA < 0.15), a further increase in BSA concentration led to the breakdown of the complex as in mechanism I: [formula: see text] At higher Cu2+ concentrations (nCu/nAA > 0.15), a further increase in BSA concentration led to the formation of non-stoichiometric polycomplexes (mechanism II): [formula: see text] The immunogenic properties of ternary mixtures of BSA-Cu(2+)-PAA were investigated and the relationship between immunogenicity and complex formation in solution was analyzed. The addition of Cu2+ to solutions of PAA with BSA gave rise to a considerable increase in BSA-specific immunogenicity. Data obtained from the analysis of the immunogenicity of BSA-Cu(2+)-PAA mixtures formed using different ratios of the components suggested that (1) the highest immunogenic activity is exhibited by stable ternary complexs, and (2) immunoactive polyelectrolyte complexes have a non-stoichiometric composition. We thus propose a novel method, based on Cu2+ mediated complex formation, to enhance protein-specific antibody responses.

Immune response to 17 beta-estradiol in polyelectrolyte complex: antigen specificity and affinity of hybridoma clones.

Complex formation between synthetic polyelectrolytes (PE) [poly-4-vinyl-N-ethyl (cetyl), pyridine bromides-PVP(R2, R16)], bovine serum albumin (BSA), or 17 beta-estradiol-BSA conjugate (BSA.E) was studied in neutral water. Weakly water-soluble (colloidal) complex was formed upon addition of BSA.E to PVP (R2, R16) solution at pH 7. A nonrandom distribution of the protein molecules between the coils of polycations and self-assembly in the nonstochiometric polycomplex particles took place. The immunogenic properties of PVP (R2, R16)-BSA.E polycomplex were investigated and the specificities of produced antibodies analyzed. 17 beta-Estradiol introduced in polyelectrolyte complexes (PE-BSA) was found to invoke considerable increases in the steroid-specific immunoresponse. However, a comparative study of immunogenic activity of polycomplexes versus BSA.E+incomplete Freund's adjuvant (IFA) mixtures revealed some differences in regards to the specificity of antibody production. In contrast to IFA+BSA.E systems, polycomplexes were able to generate estradiol-as well as BSA-specific antibodies. Such a carrier-directed response may be determined by increase in immunogenicity of weak antigenic determinants and/or by the exposure of internally located determinants upon complex formation with polyelectrolytes. Fusions following the two different immunization procedures resulted in the growth of comparable numbers of estradiol-specific monoclonal antibodies with apparently similar antigen affinities. Thus, immunizations using antigens in PEC appear to provide an efficient alternative to IFA.

[Polymer-metal complexes of the Mycobacterium protein antigen prevent the dissemination of postvaccinal infection in the T-deficiency state]. / Polimermetallicheskie kompleksy belkovogo antigena mikobakterii predotvrashchaiut disseminatsiiu postvaktsinal'noi infektsii pri T-defitsitnom sostoianii.

Primary obtained triple highly immunogenetic complex consists of nonimmunogenic protein antigen, isolated from the BCG, nontoxic polyelectrolyte and bivalent cuprum ions. Immunization with this complex provides high immunoprotection in the experimented B mice (T-deficit mice) from lethal dissemination BCG. Inoculation of single components of the complex has no protection. Result of these experiments suggest that antigen-polyelectrolyte complexes obtained on the basis of nonimmunogenic protein fraction of the tuberculosis bacilli show the properties of a thymus-independent immunization preparations, and provide immune defense in the T-cell deficit of the immune system.

[The effect of structural-chemical characteristics of water-soluble polyelectrolyte complexes of ovalbumin on their immunological properties]. / O vliianii strukturno-khimicheskikh osobennostei vodorastvorimykh poliélektrolitnykh kompleksov oval'bumina na ikh immunologicheskie svoistva.

Immunogenicity of soluble protein antigens in the complexes with synthetic polyions may be regarded as depending both on the nature of polymer carrier and the structure of the protein-polyelectrolyte complex. The immunogenicity of stable soluble complexes of ovalbumin (OA) with polycation - quaternized poly-4-vinylpyridine (C-1) and copolymer of acrylic acid and 2-methyl-5-vinylpyridine (C-2) have been evaluated. Immunization of mice by C-1 have induced a vigorous formation of the anti-OA IgG antibodies and IgE homocytotropic antibodies, while immunogenicity of OA in C-2 was comparable with that of OA alone. The analysis of the structural-chemical features of the complexes investigated has shown that enhanced immunogenicity of C-1 may be due to (1) the non-homogeneous distribution of protein globulae among polycation macromolecules and to (2) the formation of complex with an asymmetrical structure, to (3) the high ability of C-1 to adsorb on a surface of the lymphoid cells and to induce a formation of intercellular aggregates. An enhancing of a stability and a size of C-2 in the presence of Cu2+ shows no influence on a immunogenicity of OA. An immunogenicity of both types of complexes does not depend upon the access of determinants of OA to antibodies so far as it has been shown that complex formation in both cases are not accompanied by an alteration of antigenicity and allergenicity of OA.

Polymer-metal complexes of protein antigens--new highly effective immunogens.

The mechanism of interaction of the copolymers N-vinylpyrrolidone-acrylic acid and N-vinylpyrrolidone-maleic anhydride with bovine serum albumin, influenza virus total surface antigen (haemagglutinin and neuraminidase), and the BCG protein fraction in the presence of divalent copper ions was investigated. Novel water-soluble triple polymer-metal complexes of the above protein antigens were formed. These complexes showed high immunogenicity and conferred high levels of immunological protection. Study of the replication of pathogenic influenza A virus in animal lungs showed that, in mice immunised with the triple complex containing surface glycoprotein influenza virus A antigens, reproduction of the homologous virus was sharply inhibited, and immunisation of B mice, exhibiting pronounced T-cell deficiency, with complexes containing the BCG protein fraction ensured development of a high level of protection with respect to BCG infection.

[Relation of the immunogenicity of artificial antigens to the number of polyelectrolyte-bound protein molecules]. / Zavisimost' immunogennosti iskusstvennykh antigenov ot kolichestva sviazannykh poliélektrolitom belkovykh molekul.

Complexes and covalent conjugates of protein antigens with polyelectrolytes of different molecular mass have been synthesised. The structure and composition of the resulting water-soluble complex particles were determined. Artificial antigen immunogenicity was shown to depend on the amount of protein molecules complexed with polyelectrolytes. Direct correlation between immunostimulating activity of the polymer-carrier, immunogenicity of complex antigens and size-dependent capacity of the polymer molecule to aggregate protein globules has been established.

[Immunogenicity of a conjugate of bovine serum albumin with polyacrylic acid]. / Immunogennost' kon"iugata bych'ego syvorotochnogo al'bumina s poliakrilovoi kislotoi.

The conjugate of bovine serum albumin (BSA) with polyacrylic acid, in contrast to pure BSA and the mixture of BSA with polymer, induces a pronounced primary humoral response. The injection of the conjugate in Freund's complete adjuvant leads to considerable immune response to BSA, 30-40 and 100-130 times more intensive than that produced, respectively, by the injection of the conjugate alone or BSA in the adjuvant.

[Modification of the immune response by using an antigen bound to synthetic polyelectrolytes]. / Modifikatsiia immunnogo otveta pri pomoshchi antigena, sviazannogo s neprirodnymi poliélektrolitami.

The immunogenic properties of the soluble complexes of bovine serum albumin (BSA) and synthetic polyelectrolytes were studied. The polyelectrolytes used in these complexes were 4-vinyl-N-ethylpyridinium bromide and 4-vinyl-N-cetylpyridinium bromide (complex I), 4-vinylpyridine and 4-vinyl-N-acetylpyridinium bromide (complex II). C57BL mice were immunized with different doses of BSA, complexes I and II introduced intraperitoneally in a single injection, and the number of plaque-forming cells (PFC) in the spleen was determined by modified Jerne's test with the use of BSA-covered sheep red blood cells. The above complexes were shown to stimulate the production of PFC against BSA 50-100 times more intensively than pure BSA. The mixtures of BSA with the above-mentioned polyelectrolytes stimulated PFC production to a considerably lesser extent. Thus, the polymeric part of these conjugates was not an antigen, but served as a carrier inducing pronounced immune response to the antigenic (protein) part of the complex.¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿

[Mechanism of heparin binding by polycations in the presence of serum proteins]. / O mekhanizme sviazyvaniia geparina polikationami na fone syvorotochnykh belkov.

Mechanism of heparin interaction with the soluble complexes polyacrylamide (PA)--protein fractions of the whole serum has been examined. Addition of heparin to a mixture of the soluble complexes serum proteins (SP)--PA gives rise to the macromolecular reaction of protein replacement by negatively charged polysaccharide (heparin) according to the following scheme: (PA)--SP complex) plus heparin in equilibrium or formed from (PA--heparin complex) pus SP. Succession has been studied of heparin removal of different proteins from their polycation-including soluble complexes.

[Competitive interactions of serum protein fractions during complex formation with polycations]. / O konkurentnykh vzaimootnosheniiakh fraktsii belkov syborotki pri obrazovanii kompleksov s polikationom.

Interaction of gamma-globulin with quaternized poly-4-vinylpyridine in water solutions at pH 7 has been studied. Formation of soluble stable cooperative complexes has been observed in a wide range of component ratios. Protein globules are distributed unevenly between adsorbing polycations. Soluble complexes are rod-like particles assembled from the globules which are stabilized by polycation chains. Complex formation in the system gamma-G + PE is similar to that in the system BSA + PE. Competitive interaction of serum protein fractions was studied at the interacting with polycation. It has been shown that selectivity at binding protein fractions is observed in both artificially prepared systems (BSA + gamma-G, beta1-G + gamma-G, BSA + gamma-G + beta1-G), and in serum and whole blood. In those ratios where uneven distribution of protein molecules is observed the soluble complexes protein-PE are formed by separate distribution of individual proteins at the matrix. Decrease of PE concentration in the systems results in the formation of a soluble complex of mixed composition. When an insoluble complex is formed in the system serum-PE selective sorbtion of beta 2-globulin fractions is observed. The reasons for the selective sorbtion of various protein fractions are described, structural models of the soluble complexes protein-PE are suggested.