[Identification by enzyme immunoassay of escape mutants S143L and G145R of hepatitis B virus (Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus)].

INTRODUCTION: The achievement of the goal of the World Health Organization to eliminate viral hepatitis B by 2030 seems to be problematic partly due to the presence of escape mutants of its etiological agent, hepatitis B virus (HBV) (<i>Hepadnaviridae: Orthohepadnavirus: Hepatitis B virus</i>), that are spreading mainly in the risk groups. Specific routine diagnostic assays aimed at identification of HBV escape mutants do not exist.The study aimed the evaluation of the serological fingerprinting method adapted for routine detection of escape mutations in 143 and 145 aa positions of HBV surface antigen (HBsAg). MATERIAL AND METHODS: HBV DNA from 56 samples of HBsAg-positive blood sera obtained from donors, chronic HBsAg carriers and oncohematology patients has been sequenced. After the identification of mutations in HBsAg, the samples were tested in the enzyme-linked immunosorbent assay (ELISA) kit «Hepastrip-mutant-3K¼. RESULTS AND DISCUSSION: Escape mutations were detected mainly in patients with hematologic malignancies. Substitutions in 143 and 145 aa were found in 10.81% and in 8.11% of such patients, respectively. The G145R mutation was recognized using ELISA kit in almost all cases. The kit specifically recognized the S143L substitution in contrast to the S143T variant. The presence of neighbor mutation D144E can be assumed due to it special serological fingerprint. CONCLUSION: ELISA-based detection of escape mutations S143L, D144E and G145R can be used for routine diagnostics, especially in the risk groups. The diagnostic parameters of the kit can be refined in additional studies. This immunoassay and methodology are applicable for the development and quality control of vaccines against escape mutants.

[Detection of Epstein-Barr virus genome in oral cavity squamous cell carcinoma samples of russian patients.]

INTRODUCTION: Oral cavity squamous cell carcinoma (OC-SCC) is the most common and aggressive malignancy of the oral cavity. Recent studies have revealed infections with human papilloma virus (HPV) as an additional risk factor for oral squamous cell carcinoma development, while distinguished role of Epstein-Barr virus (EBV) remains still uncertain. However, the evidence for association between virus infection and risk of oral squamous cell carcinoma is controversially and varies significantly by geographic regions and race. PURPOSE: The aim of the present study was to elucidate the prevalence of HPV and EBV in OC-SCC samples of Russian patients from Moscow region. MATERIAL AND METHODS: We investigated fresh-frozen tumor tissue fragments obtained from 11 patients with OC-SCC. DNA was extracted and the viral genome was examined by quantitative PCR assays with highrisk type-specific HPV and EBV specific markers followed by sequencing-based analysis. RESULTS: No HPV infection in analyzed OC-SCC samples was observed, while EBV was identified in 70.0% (7/10) of patients. Further based on Q-PCR amplification of the EBV targets including BamHI-W, EBNA1 and C-terminal fragment of LMP1 gene, EBV infection and measurement of virus load in the tumor samples was assessed. Sequencing LMP1-positive products revealed that the most samples (5/6) contained variants LMP1 with Cao deletion characterized by an increased transforming potential. CONCLUSION: These data suggest that prevalence of EBV infections is common and may influence cancer development, although detected LMP1 variants of EBV are not necessarily associated with the pathogenesis of OC-SCC. Further studies are necessary to determine the potential role of EBV and its possible importance as an infection factor in OC-SCC.

Pretreatment with Antiviral Preparation Kagocel Normalizes the Content of Bone Marrow Multipotent Stromal Cells and TNFα in Blood Serum of CBA Mice Disturbed by Administration of S. typhimurium Antigen Complex In Vivo and Maintains High Concentration of IL-10 and Th1 Cytokines.

Pretreatment with the active substance of antiviral preparation Kagocel, inductor of type I endogenous IFN, in a daily therapeutic dose (30 µg/mouse) 3 h prior to administration of S. typhimurium antigens to CBA mice reduced the number of bone marrow multipotent stromal cell (significantly increased by 3.2 times on the next day after antigen injection) to the initial level. Thus, activation of the stromal tissue induced by administration of the bacterial antigen was blocked. In addition, preliminary administration of Kagocel modulated the cytokine profile of the blood serum affected by S. typhimurium antigens: reduced 1.6-fold elevated concentration a proinflammatory cytokine TNFα to the control level (in 4 h after antigen injection) and maintained this level in 20 h after antigen administration. Kagocel also maintained the concentration of anti-inflammatory cytokine IL-10 at the level surpassing the normal by 1.6 times and high concentrations of Th1 cytokines (IL-2, IFNγ, and IL-12). These results suggest that Kagocel can reduce the immune response to bacterial antigens (similar to type I IFN [7]), which can contribute to its therapeutic and preventive effects in addition to its well documented antiviral activity and then this preparation can be used for the therapy of diseases accompanied by excessive or chronic inflammation.

[A comparative characteristic of antigenic properties of recombinant and native hbs-antigens with G145R mutation and evaluation of their immunogenicity].

BACKGROUND: One of the important reasons for spreading of hepatitis B virus (HBV) under conditions of vaccinepressure is emergence of escape mutations. Prevalent G145R mutation in S-gene leads to the most expressed changes of serological properties of HBV. Consequently, HBsAg is modifed so thoroughly that it cannot be recognized by the majority of anti-HBs. Mutant G145R also differs from a wild type HBsAg by its immunogenic properties. At present, the relevance of enhancement of hepatitis B vaccine in view of mutant virus variants has been recognized. OBJECTIVES: a comparative study of antigenic and immunogenic properties of native and recombinant G145R mutants and an estimation of possibility for developing antigenic component of hepatitis B vaccine with G145R mutation in HBsAg. METHODS: antigenic properties of recombinant HBsAg with G145R mutation were compared with each other and with native mutants by serological fngerprinting method. Then, BALB/c mice and sheep were immunized with selected recombinant antigen under different protocols. Titers of antibodies specifc to wild type or mutant G145R type of HBsAg in sera of immunized animals were measured. RESULTS: it was found that not all the recombinant HBsAg variants with G145R substitution have the same antigenic properties as native HBsAg with similar mutation. Recombinant HBsAg selected according to the principle of antigenic similarity possesses immunogenicity in mice and sheep causing the production of antibodies reacting with native wild and mutant type HBsAg. It was shown that mutant antigen is less immunogenic, requires larger doses and more time for the development of immune response; however, it is capable of causing an antibody level comparable with wild type antigen. CONCLUSIONS: preliminary selection of recombinant HBsAg containing G145R mutation with antigenic and immunogenic properties similar to the native analogue creates the basis for development of a specifc component of hepatitis B vaccine with escape mutation G145R in HBsAg.

MORPHOLOGICAL ANALYSIS OF HEPATITIS B VIRUS WITH ESCAPE MUTATIONS IN S-gene G145R AND S143L.

BACKGROUND: In terms of serological properties and immunization, the wild type of HBsAg HBV and its G145R mutant behave as different antigens. This testifies to serious structural changes, which presumably could have a significant impact on the morphogenesis of virions and subviral particles. Nevertheless, morphological and ultrastructural investigations of HBV with G145R mutation have not been carried yet. OBJECTIVES: Research of structural and morphological organization of HBV in the presence of the G145R escape mutation. METHODS: Studies of sera, purified viruses and recombinant HBsAg were carried out by transmission electron microscopy by the method of negative staining and indirect reaction of immunelabeling using monoclonal antibodies of different specificity. Specimens of wild type HBV and HBV with S143L mutation obtained in an identical manner were used as the control. RESULTS: The presence of typical virus particles of HBV was shown in the specimens of wild strain and HBV with S143L mutation. Specimens of HBV with G145R mutation were characterized by expressed morphological heterogeneity. In the initial serum and in the specimen of purified virus containing G145R mutant, large oval particles 60-70 nm and up to 200 nm in size, respectively, were found. The presence of antigen structures of HBV in all heterogeneous forms was confirmed. It was shown that forming of subviral particles in the process of expression of the recombinant HBsAg with G145R mutation depends on conditions of expression and purification of the protein. They can vary from well-formed circular and oval particles to practically unstructured fine-grained masses. CONCLUSION: Direct data on the impact of G145R escape-mutation in S-gene, in contrast to S143L mutation, on the morphogenesis of virions and subviral particles of HBV were obtained.

Effects of Kagocel® on the Counts of Multipotent Stromal Cells, Expression of Cytokine Genes in Primary Cultures of Bone Marrow Stromal Cells, and Serum Cytokine Concentrations in CBA Mice.

The efficiency of cloning of bone marrow multipotent stromal cells (ECF-MSC) from CBA mice and the MSC counts in the femoral bone increased 24 h after a single in vivo (but not in vitro) injection of kagocel (active substance of antiviral drug Kagocel (®) ) 1.4 times (in response to 50-80 µg) and 4.6 times (in response to 250 µg). The maximum increase of ECF-MSC in response to 50 µg per mouse was detected just 1 h after Kagocel injection to intact mice and to mice previously receiving the drug for 3 days (2 and 1.7 times, respectively). The increase of ECF-MSC was 3-fold less intense in response to oral Kagocel in a dose of 250 µg/mouse vs. intraperitoneal Kagocel, ECF-MSC corresponding to its level in response to oral Poly (I:C). In vivo Kagocel led to emergence of proinflammatory cytokine IFN-γ, IL-1ß, and IL-8 mRNA in primary cultures of bone marrow stromal cells. Serum concentrations of IL-2, IL-5, IL-10, GM-CSF, IFN-γ, TNF-α, IL-4, and IL-12 increased 1.5 and 2 times just 1 h after Kagocel injection in doses of 30-50 and 250 µg, respectively, to intact mice and to animals previously treated with the drug for 3 days. The cytokine concentrations normalized after 3 h and increased again after 24 h, though did not reach the levels recorded 1 h after the drug injection. These data indicated that the therapeutic and preventive effects of Kagocel, together with its previously demonstrated stimulation of α- and ß-interferon production during several days, could be due to the capacity of this drug to increase the bone marrow ECF-MSC, serum cytokine concentrations, and induce the expression of proinflammatory cytokine genes in the bone marrow stromal cells 1 h after its injection.

[IMMUNOPATHOGENESIS OF OCCULT INFECTION CAUSED BY HEPATITIS B VIRUS].

The concept of occult infection caused by hepatitis B virus (HBV) is determined as the presence of HBV DNA in blood sera or liver with the absence of detectable HBsAg. The actuality of this problem is associated with the fact, that occult hepatitis B (OHB) can be transmitted during hemotransfusions, cause reactivation of chronic hepatitis B in immune compromised individuals, facilitate development of liver cirrhosis and hepatocellular carcinoma. Several different hypotheses of OHB immunopathogenesis have been proposed, including a low number of copies of HBV DNA, altered immune response of the macroorganism, genetic variability of the S gene, integration of viral DNA into host genome, infection of mononuclear cells of peripheral blood, presence of immune complexes that hide HBsAg, and interference by other viruses such as HCV and HIV. Molecular mechanisms of HBV virus in HBsAg-negative individuals are not fully understood, however, viral mutations seem a very significant factor. Approaches of OHB prophylaxis including use of a polyvalent vaccine, that allows vaccination against wild and mutant HBV viruses, are examined.

[Multidirectional effect of MIF and sodium polyprenyl phosphate on the course of experimental flavivirus infection in mice].

AIM: Study of macrophage migration inhibiting factor (MIF) effect after intracerebral administration on the course of experimental infection induced in mice by tick borne encephalitis virus (TEV), and study of sodium polyprenyl phosphate (PPP) and/or antibodies against MIF on the course of this infection against the background of MIF administration. MATERIALS AND METHODS: Phosprenil preparation was used as a source of PPP. PPP was administered intracerebrally. MIF--human recombinant (R&D, USA), mice--Balb/c line. RESULTS: In the sera of mice infected with TEV, MIF production stimulation was detected at days 8 through 10 after the infection--against the background of clinical signs presentation of tick borne encephalitis (TE). Administration of PPP to infected mice, on the contrary, resulted in MIF production suppression at the specified period. After administration of 20 ng of MIF to mice, lethality increased by 40% and average life span decreased by 2.3 days. Thus, MIF at high doses caused an increase of infection course severity, induced by TEV in mice, and administration of 60 microg of PPP resulted in the protection from infection in 100% of cases. Intracerebral administrationto mice of antibodies against MIF resulted in a decrease of lethality indicator up to 26% as compared with control and an increase of averagelife span by 5.5 days. During simultaneous administration into the brain of infected mice of MIF, PPP and antibodies against MIF, prevention of MIF-induced increase of TE course severity was registered. CONCLUSION: The data obtained allow to conclude that MIF may serve as an indicator of TE course severity, and possible prognostic indicator of meningo-encephalitic form development in humans.

[Role of macrophage migration inhibitory factor in systemic inflammatory response and development of sepsis].

I.I Mechnikov was the first who characterized phagocytosis as cellular defense mechanism. Infiltration of infectious process focus with phagocytes and subsequent activation of these cells is a fundamental defense reaction of the organism. However inflammation may be destructively dangerous if inflammatory response prolongates and/or generalization of the process leading to death of the host develops. The main trigger mechanisms in the pathogenesis of systemic inflammatory response and sepsis are release of bacterial endo- and exotoxins as well as hyperproduction of proinflammatory cytokines. Macrophage migration inhibitory factor (MIF) has exceptional multifunctionality and significant potential for activation of inflammatory system by various mechanisms acting as proinflammatory cytokine, hormone-contaregulator of immunosuppressive effect of corticosteroids and regulator of glucose metabolism. Data about the role of MIF as a crucially dangerous factor in pathogenesis of systemic inflammatory response obtained on experimental models of sepsis as well as about the efficacy of anti-MIF therapy were discussed, specific molecular mechanisms were analyzed. Prognostic value of high blood concentration of MIF during septic complications in clinic situations was assessed. In general, existing data on key role of MIF in sepsis pathogenesis show that MIF is one of the most promising targets for development of new strategies of immunotherapy for this life-threatening pathology.

[Assessment of sensitivity of commercial test-systems for HBsAg immunodetection according to their ability to detect HBsAg-mutants of hepatitis B virus].

Comparative study of widely distributed on Russian market commercial test-systems for HBsAg detection by enzyme immunoassay was performed. Panel of serum samples containing mutant forms of HBsAg was developed for the study. It showed that only 2 out of 7 studied test-systems are able to detect mutant forms of HBsAg with the same sensitivity as "wild-type" forms of HBsAg. Test-systems based only on monoclonal antibodies did not detect HBsAg with G145R substitution in concentration 5 ng/ml. The study confirms the relevance of problem of HBsAg-mutants detection for hepatitis B immunodiagnostics.

Effect of macrophage migration inhibition factor on the content of stromal precursor cells in mouse bone marrow and efficiency of bone marrow precursor cell cloning in vitro.

The content of stromal precursor cells in the bone marrow of mice decreased 2-5.7 times 24 h after injection of macrophage migration inhibition factor in doses of 0.1-50 ng/kg, this reduction depending on the dose of inhibition factor. The content of precursor cells in the bone marrow of mice increased 2-fold 24 h after injection of S. typhimurium bacterial mass. One day after injection of S. typhimurium bacterial mass, the count of precursor cells in mouse spleen was 7-fold higher than 24 h after injection of macrophage migration inhibition factor. The efficiency of cloning of mouse bone marrow stromal precursor cells in vitro was suppressed 1.7-2.8 times in the presence of macrophage migration inhibition factor in doses of 0.1 to 50 ng/ml culture medium. The effect of cloning inhibition was preserved, if macrophage migration inhibition factor was added to the culture medium after 2 days of bone marrow cell culturing. In general, macrophage migration inhibition factor inhibits stromal precursor cells in vivo and in vitro. The data also indicate that macrophage migration inhibition factor is not responsible for rapid and sharp increase in the count of stromal precursor cells after immunization of animals.

[Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens].

Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.

[Algorithm of serologic screening and assessment of prevalence of serologically meaningful mutations of HBsAg in hepatitis B virus carriers].

Algorithm of serologic screening for HBsAg-mutants in hepatitis B virus (HBV) carriers with high level of HBsAg was developed which is based on the detection of defects of interactions of serum HBsAg with monoclonal anti-HBs realizing as a decrease of ELISA sensitivity in 10 times or more during serial 10-fold dilutions. During 1st stage commercial test-systems based on monoclonal antibodies was used to select serum samples with discrepancy of test results. During 2nd stage HBsAg contained in selected sera was analyzed by the panel of monoclonal and polyclonal anti-HBs conjugates using decrease in ELISA sensitivity as a criterion. Serum samples from 2510 chronic carriers of HBV with high level of HBsAg were studied. 19 samples with discrepant results were found. Subsequent characterization of HBsAg with panel of 11 monoclonal and 1 polyclonal conjugates allowed to distinguish groups of sera with specific serologic "portraits". Atypical features of HBsAg were confirmed by genotyping 9 of 19 samples. Analysis of primary nucleotide sequence revealed serologically meaningful mutations in S-gene of HBV in all 9 isolates: 3 of them contained substitution mutation G145R, 5--S143L, and one--T143M. Distribution of mutations in HBsAg corresponded with specific serologic "portraits". Prevalence of HBsAg mutations in HBV carriers with high level of HBsAg was assessed for the first time: prevalence of G145R, S143L/T143M mutations, and all serologically atypical variants was 0.12%, 0.24%, and 0.76% respectively. Developed algorithm was proposed for epidemiologic monitoring of HBsAg-mutants of HBVand control of diagnostic test-systems.

[Cell-cooperation integrated test for the detection of immune reactivity to Shiga-toxin in patients with acute intestine infection]. / Integrativnyi test kletochnoi kooperatsii dlia vyiavleniia immunnoi reaktivnosti k shiga-toksinu u bol'nykh ostrymi kishechnymi infektsiiami.

Described are the results of approbation of the cell-migration screening test reflecting the outcome of cooperation between T- and B-cells and macrophages. It was shown, in adults and children with intestine infection, to be a highly-effective tool for the detection of suppressed immune response during early disease stages at stimulation in vitro by Shiga toxin at nano- and picogram concentration; it can also be used for the evaluation of the shaping specific anti-Shiga-toxic immune response. The parameters of the migration activity of peripheral-blood leucocytes at exacerbation and convalescence were demonstrated to correlate with the age of sick children and with the severity of intestine infection as well as with the level of a Shiga-like toxin detected in coprofilters and circulating immune complexes.

[Evaluation of the system for controlling the quality of the HBsAg detection in the system of screening laboratories]. / Otsenka sistemy kontrolia kachestva detektsii HBsAg v seti skriningovykh laboratorii.

The two-stage control system, ensuring the high quality of serological investigations in the network of screening laboratories in Moscow was developed and introduced into practice. At the first stage the entry control of the quality of the test system for the detection of HbsAg, coming to the screening laboratories, is made in the reference laboratory with the use of specially developed "representative" panels, as well as the test systems for comparison ("reference" test systems). Then "minipanels" are formed from specimens included into the "representative" panels, which are used for the evaluation of the quality of laboratory investigations made in the screening laboratories. The high quality of such system for controlling the quality of the detection of HBsAg in the screening laboratories of Moscow is shown.

[Reverse correlation between natural resistance of mice to transplanted leukemia EL-4 and rectal temperature]. / Obratnaia korreliatsiia mezhdu estestvennoi rezistentnost'iu myshei k perevivaemomu leikozu EL-4 i rektal'noi temperaturoi.

Mice F1 (CBA x C57Bl) life span after transplantation of standard number of leukemia EL-4 cells at different clock hours was evaluated. At the same time, as injection was done, rectal temperature of mouse was measured. Both parameters showed circadian trend. Mice life span was in negative correlation with rectal temperature. So it is possible to consider temperature rhythm as marker rhythm of natural antitumor resistance.

[Serum factors regulating macrophage migration in chronic cicatricial stenosis of larynx and trachea in children]. / Sootnoshenie syvorotochnykh factorov, reguliruiushchikh migratsiiu makrofagov pri khronisheskom rubtsovom stenoze gortani i trakhei u detei.

Many children with chronic cicatricial stenosis of the larynx and trachea showed a high activity of the factor stimulating macrophage migration in vitro (MSF). The high MSF level is typical of patients with large scars in tissues rich in cellular elements. The MSF predominance was to a certain extent correlated with abnormal responses to surgical intervention. This was indicated by poor results of surgical treatment if the MSF was low before and immediately after it. With normal MSF the treatment was effective in at least 50% of cases. The therapeutic effectiveness was correlated with MSF variations at an early postoperative stage.

[Immunologic mechanisms of the development of nonspecific aortoarteritis]. / Immunologicheskie mekhanizmy razvitiia nespetsificheskogo aortoarteriita.

It has been shown that patients with nonspecific aortoarteritis undergoing acute and subacute stages of the process demonstrate sensitization to collagen, types I and III. The activity of inflammation was discovered to affect the disease progression and involvement of new arterial areas. Marked suppression of leukocyte migration in response to collagen, types I and III, in patients with nonspecific aortoarteritis points to a high probability of further disease progression.

[The viroimmunotest for mass determinations of nanogram amounts of antigens]. / Viroimmunotest dlia massovykh opredelenii nanogrammovykh kolichestv antigena.

Simple highly sensitive screening variant of viral immunoassay for detection of nanogram quantity of antigen is proposed. The antigen linked with antibodies adsorbed on polystyrene 96-well plates was revealed by conjugate of Fab' fragments of antibodies with phage phi X174. The concentration of the antigen could be detected qualitatively and quantitatively (from 0.3 ng/ml to 1000 ng/ml).

[Determination of lymphokine-induced cytolytic activity of macrophages by measuring the 3H-thymidine residue in prelabeled tumor cells]. / Opredelenie indutsirovannoi limfokinom tsitoliticheskoi aktivnosti makrofagov po ostatku 3H-timidina v predvaritel'no mechennykh kletkakh opukholi.

A technique of macrophage-activating factors (MAF) detection by the in vitro determination of macrophage (Mph) antitumour cytolytic activity by 3H-thymidine residue in the pre-labelled neoplastic target cells (TC) is suggested. Peptone-induced Mph were cultivated for 20-22 hours in the presence of crude supernatants from concanavalin-A-stimulated spleen cells or from the secondary mixed lymphocyte culture. 3H-thymidine-labelled cells of mastocytoma P815 were then added to the washed Mph. The lysis was measured in 48 hours according to the isotope remainder in the non-acid-soluble fraction. The optimal conditions for MAF detection have been selected. Parallel MAF testing with this particular method and with a standard technique using labelled 51Cr TC made it possible to conclude that the method suggested is more sensitive because it permits a combined cultivation of Mph and TC for a relatively long time period.