Detection of influenza viruses through selective adsorption and detection of the M-protein antigen.

A model system has been developed which permits rapid detection of influenza viruses through targeting of the M (membrane or matrix)-protein; a type-specific antigen, in an enzyme-linked immunosorbent assay system. This technique exploits the hydrophobic properties of M-protein; the M-protein is selectively and rapidly adsorbed to polystyrene surfaces even in the presence of a 5000-fold excess of bovine serum albumin. Hyperimmune antiserum prepared to purified M-protein is used as the detecting reagent. All type A influenza viruses could be detected by this technique, type B influenza viruses reacted to a slight extent and Sendai virus (parainfluenza virus, type 1) did not react. Virus could be detected to levels as low as 3 ng. Purification of M-protein and preparation of hyperimmune sera from other related virus groups, such as type B influenza viruses, paramyxoviruses and rhabdoviruses should permit detection of these agents by a similar technique.

[Myxovirus structural investigations by spin probe methods. II. Influence of rimantadine on the structure of viral and artificial lipid membranes]. / Issledovanie struktury miksovirusov metodom spinovykh zondov. II. Vliianie rimantadina na strukturu virusnykh i iskusstvenno sformirovannykh lipidnykh membran.

The lipid membrane structure of two myxoviruses (influenza and Sendai viruses), red blood cells and liposomes (prepared from total lipids of Sendai virus) have been investigated in the presence of rimantadine by means of two spin probes C5 and C6--the iminoxyl derivatives of stearic acids. Rimantadine was shown to penetrate the lipid membranes of myxoviruses and red blood sells and change the structure of the lipid phase no less than 0.8 nm apart from the viral surface. In the depth of 2.2 nm the structure of these lipid membranes remains unchanged after addition of rimantadine. Rimantadine also displaces the phase transition points of cell and viral lipid membranes to lower temperatures. Rimantadine does not change the lipid bilayer structure of liposomes 0.8 nm apart from its surface. These finding allow to suggest that rimantadine affects the cell and viral lipoprotein membranes.

[The effect of detergents on the physicochemical and immunochemical properties of the isolated M1 protein of the influenza virus]. / Vliianie detergentov na fiziko-khimicheskie i immunokhimicheskie svoistva izolirovannogo belka M1 virusa grippa.

The degree of solubility of influenza virus protein M1 preparations isolated from virions by acid chloroform-methanol extraction was studied under the effect of a wide spectrum of detergents of different origin. The same detergents were used for solution of a lipid comprising a part of artificially formed liposomes. Only some of the detergents used (sodium dodecyl sulfate, SDS, triton X-100, and disintegron-B) were shown to be optimal for solution of both influenza virus protein M1 and lipid. The degree of effect on the immunochemical properties of protein ML isolated from influenza virus virion of the above-mentioned detergents optimal for solution was also studied. For this purpose, a panel of 18 monoclonal antibodies with different determinant specificity to protein M1 was used. Two of the three detergents (SDS and disintegron-B) were shown not to change the antigenic profile of protein M1. The immunochemical properties of protein M1 of influenza virus isolated from virions by two methods: chloroform-methanol extraction and preparative polyacryl amide gel electrophoresis, were studied. These two methods of protein M1 isolation were shown not to alter its immunochemical properties.

[Enhanced immunogenicity of influenza virus surface proteins when incorporated into liposomes]. / Povyshenie immunogennosti poverkhnostnykh belkov virusa grippa pri ikh vkliuchenii v liposomy.

A method for incorporation of influenza virus surface proteins into monolayer liposomes has been developed on the basis of removal of dialysed non-ionic detergent (beta-octyl glucoside) from a mixture of viral proteins and lipids. The resulting lipid-protein complexes (virosomes) are similar to intact virus particles by their morphological parameters. The immunogenicity of hemagglutinin incorporated into virosomes was significantly higher than that in the preparation of surface proteins isolated from virions which was inoculated into mice with or without complete Freund's adjuvant. The high immunogenicity of hemagglutinin incorporated into liposomes correlated with their high hemagglutinating activity. The possibility of using virosomes as subunit influenza vaccines is discussed.

[Localization of the tryptophan residues of influenza virus matrix protein in reconstructed proteoliposomes: a fluorescent analysis]. / Lokalizatsiia ostatkov triptofana matriksnogo belka virusa grippa v rekonstruirovannykh proteoliposomakh: fliuorestsentnyi analiz.

Influenza virus matrix protein (M-protein) interaction with model phospholipid membranes, liposomes, was studied. Measuring of the effectiveness of energy transfer from M-protein triptophan residues to a fluorescent zond pyren included into the lipid phase of proteoliposomes was employed to assess the steric organization of the proteoliposome protein-lipid complex. A steric model is proposed in which M-protein molecules are located on the surface of lipid bilayer forming trimers. Analysis of pyren fluorescence proper demonstrated a strong influence of M-protein on the lipid bilayer structure: the viscosity of the lipid phase in the presence of M-protein was increased 2.3-fold.

[The spatial interrelationships between influenza virus hemagglutinin and the lipid phase in the liposomal membrane]. / Issledovanie prostranstvennykh vzaimootnoshenii mezhdu gemaggliutinom virusa grippa i lipidnoi fazoi v membrane liposom.

A scheme has been proposed demonstrating the location of tryptophan residues of hemagglutinin molecule in relation to the middle of the lipid layer 1.2 nm thick with a fluorescent probe pyrene. In the immediate proximity to it, one tryptophanyl of protein molecule is located in a hydrophobic "pocket". At a distance of 2.85 nm from the middle of the lipid zone 3 tryptophanyls are located and the remaining five at a distance over 3.6 nm. After treatment with proteolytic enzyme bromelin of the liposomes with hemagglutinin incorporated into their bilayer, the hydrophobic "anchor" of protein molecule contains one tryptophanyl which is raised by 0.3 nm and its hydrophobic environment is changed.

[Effect of remantadine and amantadine on the interaction of influenza virus proteins with model lipid membranes]. / Vliianie remantadina i amatadina na vzaimodeistvie belkov virusa grippa s model'nymi lipidnymi membranami.

The influence of adamantane derivatives (remantadine and amantadine) on the surface charge of lipid bilayer when they are adsorbed on an artificially formed bilayer lipid membrane (BLM) was studied. The presence of the final number of binding centres on the BLM surface for remantadine and amantadine molecules and the cooperative nature of interaction of these compounds with the lipid bilayer were demonstrated. The features of interaction of influenza virus proteins isolated from the virion--M protein and a mixture of surface glycoproteins--were studied. Both antiviral compounds were shown to reduce markedly M protein adsorption on the lipid bilayer and to affect negligibly the sorption properties of the surface glycoproteins. Remantadine molecules interact directly with M protein molecules reducing their hydrophobic properties and, thereby, reducing the affinity of this viral polypeptide to lipid bilayer.

Incorporation of influenza virus M-protein into liposomes.

M-protein from influenza virus vaccine was purified by sodium dodecyl sulfate-gel chromatography and incorporated into liposomes by solubilization with octylglucoside and subsequent dialysis. Liposomes containing M-protein formed a distinct population with a density of 1.22 g/ml on sucrose-gradient centrifugation, regardless of the net charge on the liposomes. Treatment of the liposomes by freeze-fracture followed by electron microscopic examination showed multilamellar structures in those liposomes without M-protein; liposomes containing M-protein were mulberry-like structures which appeared unilamellar. These studies show incorporation of M-protein into the lipid bilayer.

Influenza virus antigens conjugated with a synthetic polyelectrolyte: a novel model of vaccines.

Haemagglutinin (HA), a mixture of haemagglutinin and neuraminidase (HA + NA), and matrix (M) protein were isolated from the influenza A virus and covalently coupled to a synthetic polyelectrolyte (P). A single injection into mice of the resultant conjugates (virogates) brought about efficient stimulation of the primary immune response specific to the corresponding viral antigens. Mice immunized with virogates HA.P or (HA + NA).P were largely protected against a lethal challenge infection with homologous virus. Immunization of mice with M.P virogate containing M protein originated from a 1934 influenza strain resulted in pronounced protection against a lethal challenge infection with a 1980 strain. Virogates are discussed as a novel model of artificial vaccines.