IMMUNOHISTOCHEMICAL STUDY OF DEPIGMENTED SKIN OF VITILIGO PATIENTS.

Immunohistochemical analysis was used to study depigmented skin areas such as macular of depigmentation and skin perimakular areas in vitiligo patients. It has been shown that the cells containing melanocytic cell marker TRP1 are localized both in macular and perimakular areas. Within the macula of depigmentation all TRP1 positive cells are in close contact with the basement membrane. In perimakular areas many cells that have lost contact with the basement membrane, were localized deep in the epidermis. About 92 % of TRP1 positive perimakular cells were also vimentin positive. Vimentin positive cells were numerous in perimakular areas but missing in the macula of depigmentation. Dense groups of cells immunopositive for transcription factor Snail, known as inductor of epithelial-mesenchymal transition, were localized in perimakular areas in close proximity to the macula depigmentation border. Such cells were extremely rare within the macula of depigmentation. There is reason to assume that an intensive process, which is similar to the epithelial-mesenchymal transition, might be the cause of melanocyte death in perimakular field, and thus prevents repigmentation of depigmented areas.

Immunization with influenza A NP-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses.

Two-fold immunization of Balb/c mice with a vaccinia virus recombinant expressing the NP protein of influenza A/PR8/34 (H1N1) virus under the control of a strong synthetic promoter induced specific antibodies and protected animals against low-dose challenge by mouse-adapted heterosubtypic variants of human A/Aichi2/68 (H3N2) and avian A/Mallard/Pennsylvania/10218/84 (H5N2) influenza virus strains. The surviving immunized animals had lower anti-hemagglutinin antibody titers compared to non-immunized mice. There was no difference in viral titers in lungs of immunized and non-immunized animals that succumbed to the infection. In order to try to increase immune system presentation of NP-protein-derived peptides, and thereby increase their immunogenicity, we constructed another vaccinia-based NP-expressing recombinant containing a rapid proteolysis signal covalently bound to the NP protein. This sequence, derived from the mouse ornithine decarboxylase gene has been shown to increase degradation of various proteins. However, we found that when used as part of a recombinant NP, this signal neither increased its proteolytic degradation, nor was it more efficient in the induction of a protective response against influenza infection.

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein.

BACKGROUND: Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. RESULTS: The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. CONCLUSION: Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines.

Vaccinia virus recombinant expressing gene of tick-borne encephalitis virus non-structural NS1 protein elicits protective activity in mice.

A vaccinia virus recombinant containing the non-structural tick-borne encephalitis virus (TBEV) NS1 gene was developed. The recombinant expressed native dimeric form of the NS1 protein in infected cells and protected mice against lethal infection with TBEV.

Correction of lipid composition of the lymph and blood during immediate type hypersensitivity.

Combination therapy with hydrocortisone and norepinephrine improved resorption and transport functions of the lymphatic system during anaphylactic shock, which was accompanied by a considerable increase in the contents of total lipids, phospholipids, cholesterol, nonesterified fatty acids, and malonic dialdehyde (lipid peroxidation product) in the lymph and blood. It should be emphasized that lipid concentration in the lymph increased to a greater extent than in the blood.

Allergic alteration in the organism is associated with disturbances in lipid composition of the lymph.

Anaphylactic shock causes changes in the transport chain of lipid metabolism in the blood-tissue-lymph-blood system. During the early periods of shock, the changes in lipid composition of the lymph and blood result from increased permeability of the blood-tissue barriers, while at later terms, these processes are primarily determined by metabolic changes.

Correction of lymph circulation during immediate hypersensitivity reaction.

We measured lymph flow rate in the thoracic lymphatic duct of dogs with anaphylactic shock receiving mono- or combination therapy with norepinephrine and hydrocortisone. Intensification of lymph circulation improved resorption and transport of metabolic products from the interstitial space through lymphatic vessels and stimulated exchange processes in the blood and tissues during allergic alterations.

Genetic instability of vaccinia virus containing artificially duplicated genome regions.

A double recombinant of vaccinia virus (W-lacZ/J-tk/F) was obtained, which contains two inverted copies of the virus tk gene, separated by 45 kb: (i) the native copy located in the HindIII J fragment of the virus genome was inactivated due to insertion of E. coli lacZ gene; (ii) the second active copy was artificially inserted into the HindIII F fragment. The virus expressing both thymidine kinase and beta-galactosidase (tk+lac+ phenotype) was cloned. Due to the presence of duplicated inverted sequences of the tk gene in the virus genome extensive recombination was observed leading to genetic heterogeneity of the virus population. The population consisted mainly of the virions with the tk+lac- (77%) and tk+lac+ (23%) phenotypes. Passages in the presence of BUdR revealed minor fractions of the tk-lac+ and tk-lac- phenotypes. Structural analysis of DNA isolated from virions confirmed the genetic heterogeneity of the virus population. Nine different HindIII fragments were detected containing HindIII F, J and (or) lacZ sequences. The structure of these fragments indicates that predominantly two types of recombination events occur in the population: (i) translocation of the lacZ gene between duplicated sequences of the tk gene or displacement of lacZ by tk via intergenome and intragenome double crossing over; (ii) inversion of a 45 kb sequence in the conserved region of the genome between duplicated sequences of the tk gene due to a intragenome single crossing over.

Hepatitis B virus proteins expressed by recombinant vaccinia viruses: influence of preS2 sequence on expression surface and nucleocapsid proteins in human diploid cells.

Fifteen vaccinia virus (VV) recombinants derived from VV strains Praha, LIVP and DD (i.e. Dryvax Wyeth vaccine-derived) and expressing genes for S, preS2-S or c antigens of hepatitis B virus (HBV) were tested in monkey CV-1 cells and human diploid LEP cells. The production of infectious virus was found to be alike in all the recombinants and parental viruses as well. However, several recombinants produced markedly lesser amounts of S and preS2 antigens in LEP cells than in CV-1 cells. This reduction was independent of the parental virus used. There was, however, a relationship between the production of preS2 in CV-1 cells and the production of S and preS2 antigens in LEP cells; in general, recombinants efficiently inducing preS2 antigen formation in CV-1 cells produced markedly reduced amounts of S and preS2 antigens in LEP cells. Reduction of HBV antigen production in LEP cells was not apparent in recombinants expressing only S or c antigens of HBV, and the production of c antigen by double recombinants was not influenced by simultaneous expression of preS2 and S. The various recombinants also differed in the ratio of S:preS2 antigen formation. This difference seemed to be associated with the length of the untranslated leader sequence preceding preS2 but not with the parental virus or cell type used. The titers of antibodies against S and preS2 antigens induced in mice immunized with different recombinants differed markedly. The differences in the ratio of S:preS2 antigen production in vitro were not reflected in vivo by S:preS2 antibody ratio.

Immunogenicity of recombinant vaccinia viruses expressing hepatitis B virus surface antigen in mice.

The ability of different recombinant vaccinia viruses (RVV) expressing hepatitis B virus surface antigen (HBsAg) to induce anti-HBs and anti-vaccinia virus responses has been analyzed in mice. The RVVs tested differed with regard to the original vaccinia virus strain used as vector and the site of insertion of a foreign gene. It was found that the immunological responses to RVVs based on the WR strain were higher than those to RVVs based on the Lister strain. The immunological response was also quantitatively affected by the viral TK phenotype. The presence of the preS2 region in HBsAg gene, inserted into the RVV genome, led to the induction of anti-preS2 antibodies but decreased the antibody response to the S-portion of HBsAg.

Viable double vaccinia virus recombinants with the non-inducible phage T7 expression system.

Double vaccinia virus recombinants expressing both the T7 RNA polymerase gene, controlled by a weak early poxvirus PF promoter, and the Escherichia coli beta-galactosidase gene, controlled by the phage T7 promoter, have been obtained. The viability of the double recombinants depended on the T7 RNA polymerase expression level. If the T7 RNA polymerase gene was inserted into a recombinant already containing the beta-galactosidase gene, the efficiency of formation of the double recombinants was significantly higher compared to that for the reverse insertion order. The negative effect of the phage T7 terminator on beta-galactosidase expression in cells infected with the recombinant viruses has been shown. The dynamics and levels of beta-galactosidase formation by different vaccinia virus recombinants have been studied.

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.

[Verification of the safety, inoculability, reactogenicity and antigenic properties of a live recombinant smallpox-hepatitis B vaccine in an experiment in volunteers]. / Proverka bezopasnosti, privivaemosti, reaktogennosti i antigennykh svoistv zhivoi rekombinantnoi ospenno-gepatitnoi B-vaktsiny v opyte ne dobrovol'tsakh.

Trials of the first Soviet live recombinant smallpox-hepatitis B vaccine (SHBV) in volunteers (20 men aged 18-20 years) showed its safety, good "take"-rate, and lower reactogenicity as compared with the standard smallpox vaccine (LIVP strain). Smallpox virus-neutralizing antibodies in response to SHBV were produced as well as in response to the smallpox vaccine. Revaccination of human subjects with smallpox vaccine and SHBV 45 days after the previous vaccination resulted in antibody booster to vaccinia virus. After two inoculations of SHBV at an interval of 45 days no anti-HBsAg antibodies were found for 3 months after the last vaccination. However, even a single vaccination with SHBV induced priming to HBsAg. This could be demonstrated after inoculation of the subjects vaccinated with SHBV with one dose of plasma hepatitis vaccine. In the subjects vaccinated with SHBV antibody in response to the plasma vaccine formed more frequently and in higher titres than in those prevaccinated with smallpox vaccine or placebo.

[Detection of influenza virus A antigens by radioimmunological methods in patients' nasopharyngeal washings]. / Vyiavlenie antigenov virusov grippa A radioimmunologicheskimi metodami v nosoglotochnykh smyvakh bol'nykh.

9olid-phase radioimmunoassay (SPRIA) was used for the detection of influenza A (H3N2,H1N1) and B viruses in nasopharyngeal washings of patients admitted in January-March, 1983, to the 1st Clinical Hospital of Moscow City with acute respiratory diseases. The solid phase consisted of nitrocellulose filters and plastic plates which were coated with nasopharyngeal washings of the patients. Rabbit or horse antiviral immunoglobulins were used as antibodies. 125I-labeled protein A was the indicator system. In 61 out of 211 patients examined influenza A (H3N2) virus was detected; from 20 of the influenza A (H3N2), from 7 influenza A (H1N1) virus was isolated, but no influenza B virus was ever found. Comparisons of the results of SPRIA and IF techniques yielded similar but not identical data. Diagnostic rises of antibodies were demonstrated in 48 out of 61 patients. The lack of complete correlation between antibody rises and detection of influenza A virus antigen appears to be due to early discharge of the patients when humoral immunity had not reached its peak. The SPRIA is a highly sensitive and specific technique for influenza A virus detection in nasopharyngeal washings of the patients and may be recommended for use in properly equipped laboratories where highly specific hyperimmune sera are available. It gives an objective information on the proportion of influenza in the period of epidemic rise of ARD incidence.

[Production of noninfectious virions of the Rous sarcoma virus by virogenic hamster cells]. / Produktsiia neinfektsionnykh virionov virusa sarkomy Rausa virogennymi khomiachkovymi kletkami.

Noninfectious virions morphologically identical with avian type C virus virions were produced in Rous sarcoma virus-transformed virogenic hamster cells. The population of the virions contained the major internal protein of avian oncornavirus. It is assumed that production of defective RSV virions occurred in the cells. The major internal protein of avian oncornaviruses was found to be incorporated into the virions containing in their membrane interspecies antigens of hamster oncornavirus produced spontaneously in the system under study. Thus, phenotypic mixing of avian and animal type C viruses in mammalian cells has first been observed.

[Characteristics of the type C virus produced by Rous sarcoma virus-transformed hamster cells]. / Kharakteristika virusa tipa C, produtsiruemogo khomiachkovymi kletkami, trasformirovannymi virusom sarkomy Rausa.

Production of hamster type C virus in Rous sarcoma virus-transformed hamster cells is described. This virus preparation was shown to contain the antigen of the major inner protein of avian type C viruses (p27). The population of virions produced by such cells consists of either of virions of two types (99%--99.9% virions type C of hamster and 0.1%--1% virions the core capsule of which is formed from Rous sarcoma virus p 27) or of virions phenotypically mixed with regard to the major inner protein. The latter possibility seems less likely.

[Detection of a common antigenic determinant in the major inner protein of unrelated oncoviruses]. / Vyiavlenie obshchei antigennoi determinanty v glavnom vnutrennem belke nerodstvennykh onkovirusov.

A new heterologous system of radioimmunoassay (p24 of bovine leukemia virus--antiserum to Rous sarcoma virus) has been developed which demonstrated for the first time the existence of a common antigenic determinant in the major inner protein of unrelated oncoviruses: avian leukemia-sarcoma virus, bovine leukemia virus, mammalian type C viruses (mouse, hamster, monkey) and type D viruses (simian Mason-Pfizer virus). These data suggest a common origin of unrelated oncoviruses and open new approaches for the search of unknown agents associated with human and animal neoplastic diseases.

A new virion precipitation test for oncovirus envelope antigens which detects common antigenic determinants in mammalian type-C viruses and Mason-Pfizer monkey virus.

A method for the study of oncovirus envelope antigens was developed, bases on the precipitation of intact virions by a double antibody technique. The amount of precipitated virus was then measured as reverse transcriptase activity. The method was designated the virion precipitation test (VPT). It has been used for titration of antibodies to envelope antigens of oncoviruses. The study of envelop antigens of 11 different oncoviruses permitted their differentiation into the following groups: (1) murine type-C viruses: (2) feline type-C viruses; (3) simian type-C viruses; (4) the RD-114/BEV group; (5) Mason-Pfizer monkey virus (M-PMV); (6) bovine leukemia virus; (7) avian type-C viruses; (8) mouse mammary tumor virus. No common antigenic determinants were detected in the last three groups. Mammalian type-C viruses (RD-114, NIH-MuLV, G-MuLV) had common antigenic determinants in the envelope, as demonstrated with an anti-RD-114 serum. Mammalian type-C viruses also shared antigenic determinants with M-PMV. The relationship of type-C viruses to M-PMV decreased in the following order: RD-114--NIH-MuLV--G-MuLV. It was also shown that the endogenous xenotropic feline RD-114 virus was more closely related to xenotropic NIH-MuLV than to ecotropic G-MuLV. The nature of the common antigenic determinants, as demonstrated by VPT on the surface of mammalian type-C viruses and M-PMV, and their significance for the concept of oncovirus evolution are discussed.