Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen.

A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.

Identification of antigenic sites on three hepatitis C virus proteins using phage-displayed peptide libraries.

A novel approach to screening phage-displayed peptide libraries has been used to identify hepatitis C virus (HCV) core, NS4 and NS5 sequences, which are antigenic in humans. Two random peptide libraries were used for screening using a mixture of HCV-positive sera or individual antibodies to core, NS3, NS4, and NS5 HCV proteins affinity-purified from this mixture. Sequencing of 56 selected phage clones resulted in 28 different peptide sequences and identification of seven antigenic regions, three in the core protein (19-26, 34-49, and 73-83), three in the NS4 (1681-1693, 1712-1718, and 1726-1736) and one in the NS5 protein (2251-2260). No NS3-specific peptides were identified. The immune response to core, NS4 and NS5 proteins includes a variety of linear determinants whereas epitopes on the investigated part of NS3 protein appear to be conformation-dependent.

[Constructing the reference panel of sera with standard level of IgG-antibodies]. / Konstruirivanie standartnykh panelei syvorotok c normirovannym urovnem IgG-antitel.

Fundamentals of designing reference panels of sera for effective control of commercial test systems and immunoblotting, intended for detecting antiviral antibodies, have been developed. Reference low-titer panels of anti-IgG antibodies to HIV-a and hepatitis C virus have been designed. A reference panel contains diluted reactive sera with a standard level of IgG antibodies and native sera with undetectable level of antibodies to the major viral antigens from risk group subjects. The reactive sera of a panel contain the whole spectrum of antibodies to all principal viral antigens.

[Experimental study of the possibility of emergency prophylaxis of Bolivian hemorrhagic fever]. / Eksperimental'noe izuchenie vozmozhnosti ékstrennoi profilaktiki Boliviiskoi gemorragicheskoi likhoradki.

Describes changes in nonspecific immunity parameters (interferon, interleukin-1, tumor necrosis factor, and natural killers) in the course of experimental Bolivian fever (Machupo virus). Changes of these parameters were followed up after urgent prophylactic injections of Ridostin and specific gamma-globulin to infected animals. The possibility of treatment of experimental Machupo fever by intranasal administration of interferon inductor Ridostin has been demonstrated.

[Study of the immunobiological properties of parotitis viral strains]. / Izuchenie immunobiologichekikh svoistv shtammov virusa parotita.

The possibility of using different strains of parotitis virus (Enders, L-3, Jeryl-Leen) as antigens for enzyme immunoassay (EIA) to titer antibodies in human and animal blood sera is analyzed. Methods for preparation and purification of antigen on the basis of the said parotitis virus strains have been developed. Conditions of EIA were optimized. The sensitivity and specificity of EIA and hemagglutination inhibition test were compared.

[Study of certain indicators of immunity upon infecting CBA/Calac line mice with Lassa virus]. / Izuchenie nekotorykh pokazatelei immuniteta pri zarazhenii myshei linii CBA/Calac virusom lassa]

Some immunity parameters (interferon, tumor necrosis factor, interleukin 1, etc.) were studied in CBA/Calac mice infected with Lassa virus. The results permit a hypothesis that a pathologic inflammatory reaction is responsible for the death of animals in experimental Lassa fever. One of the components of this reaction is endogenous shock involving a manifest production of immune response mediators, such as interferon, interleukin 1. and tumor necrosis factor.

[The immunity indices in the infection of mice of different strains with the Machupo virus]. / Pokazateli immuniteta pri zarazhenii myshei razlichnykh linii virusom Machupo.

The pathomorphological patterns and the activity of serum interferon, interleukin-1, tumor necrosis factor, natural killers, and proliferative activity of lymphocytes were studied in BALB/c and C57B1/6 mice intracerebrally infected with Machupo virus. The BALB/c mice showed 100% lethality at 8-9 days after inoculation while C57B1/6 mice were found nonsusceptible to Machupo virus inoculation by this route. The pathomorphological findings at the peak of clinical manifestations in BALB/c mice revealed no organ whose functional deficiency could lead to the death of the animals. Investigations of nonspecific immunity parameters revealed a direct dependence between their high activity and susceptibility of the animals to Machupo virus infection. It is assumed that the endogenous shock due to the high activity of immune response mediators is the cause of death in Machupo virus infection.

[The ultrastructural changes in guinea pig organs during the serial passage of the Ebola virus]. / Ul'trastrukturnye izmeneniia v organakh morskikh svinok pri posledovatel'nom passirovanii virusa Ebola.

Morphological study of internal organs of guinea pigs inoculated with Ebola virus at 2-8 passages was carried out. In the course of these passages the number of infected cells and virus particles in the organs was shown to increase, and the destructive changes in organs became more pronounced. In the 1st-3rd passages Ebola virus replication was observed in macrophagal cells only but beginning from the 4th passage of virus reproduction was found also in hepatocytes, spongiocytes, and fibroblasts.

[Effect of lipid peroxidation on the insulin receptor in rat adipocytes]. / Vliianie perekisnogo okisleniia lipidov na retseptsiiu insulina adipotsitami krys.

Lipid peroxidation is studied for its effect on insulin receptors in isolated rat adipocytes. The results suggest that addition of two peroxidants (3 mM cumene hydroperoxide and 0.2 mM Fe2+) leads to malondialdehyde accumulation and binding inhibition through insulin receptors quantity and affinity decrease.