[Association of polymorphic variants of hemostatic system genes with the course of COVID-19].

INTRODUCTION:   COVID-19 is characterized by a varied clinical course. The aim of the work was to identify associations of SNPs of hemostatic system genes with COVID-19. MATERIALS AND METHODS: DNA was isolated from patients (n=117) and healthy participants (n=104). All infected patients were divided into 3 groups, depending on disease severity assessment, which was appreciated by NEWS2. Another group consisted of participants, who had asymptomatic infection in the past. Determination of SNPs of the genes FGB (-455 G/A), FII (20210 G/A), FV (1691 G/A), FVII (10976 G/A), FXIIIA1 (103 G/T), ITGA2 (807 C/T), ITGB3 (1565 T/C), SERPINE1 (-675 5G/4G) were performed by PCR using the "Genetics of Hemostasis" kit ("DNA-Technology", Russia). RESULTS: In analyzed SNPs, no significant differences were detected between the group of infected patients and healthy participants. But significant association was revealed in gene SERPINE1 (-675 5G/4G), when patient groups, differing in the disease severity, were analyzed relative to the group of participants with asymptomatic infection (p=0.0381; p=0 .0066; p=0.0009). It was found, that as COVID-19 severity scores increased, the proportion of 5G allele of gene SERPINE1 decreased, and the proportion of the 4G allele increased (p=0.005; p=0.009; p=0.0005). Similar processes were observed for genotypes 5G/5G and 4G/4G. DISCUSSION: The gene SERPINE1 (-675 5G/4G) is associated with the severity of COVID-19. CONCLUSION: For the first time, it was discovered that 5G/5G genotype of gene SERPINE1 (-675 5G/4G) can be a marker of a milder course of COVID-19, and the 4G/4G genotype as a more severe one.

[The analysis of immunoreactivity of individual B-cell epitopes of hepatitis C virus (Flaviviridae: Hepacivirus: Hepatitis С virus) NS4a antigen].

INTRODUCTION: Chronic viral hepatitis C (CHC) is a ubiquitous infectious disease, a significant limitation of which WHO attributes to the use of a new highly effective antiviral therapy. Previously, two B-cell epitopes were identified in NS4a antigen of the hepatitis C virus (HCV). It was shown that certain titers of antibodies (ABs) to the extended C-terminal epitope (1687-1718 a.a.) can predict a high probability of achieving a sustained virological response (SVR) to standard therapy with pegylated interferon-α and ribavirin.The aim of the work was to determine immunoreactivity of two B-cell epitopes (middle and C-terminal) of NS4a antigen, and to estimate a possible association of ABs to them with the achievement of SVR after standard interferon therapy and treatment with direct antiviral drugs (DAAs) daclatasvir and sofosbuvir (velpanat). MATERIALS AND METHODS: Blood serum samples of patients with CHC (n = 113), of which 55 participants received standard interferon therapy, 50 received velpanate treatment, the remaining 8 received no therapy were examined. The middle B-cell epitope (positions 24-34 a.a.) of NS4a was synthesized by the solid-phase method, while the C-terminal epitope (34-54 a.a.) was obtained using genetically engineered techniques. Enzyme immunoassay (ELISA) testing of the sera collected before treatment was performed for the two selected epitopes according to the conventional methods. RESULTS: The antibodies to the C-terminal epitope were detected significantly more frequently than those to the middle one (p = 0.01) when analyzing the blood sera of patients (n = 113). The presence of ABs to the C-terminal epitope in the serum samples of participants who completed standard interferon therapy was associated with the achievement of SVR (p = 0.0245). In the blood sera of participants who completed therapy with velpanate, an association of the presence of ABs to the C-terminal epitope with the achievement of SVR was also established (p < 0.0001). The presence of ABs to the middle B epitope was not associated with the achievement of SVR, regardless of the therapy used. DISCUSSION: The observed difference in the immunoreactivity of the two B-cell determinants may be associated with the localization of the nearest Th-epitopes, the sensitivity of NS4a antigen to proteolytic enzymes, and the peculiarities of epitope presentation by antigen-presenting cells. However, it should be noted that the immunoreactivity of the middle B-epitope is poorly studied. Although the association of ABs to the C-terminal epitope with the achievement of SVR has been shown by several scientific teams, the detailed molecular mechanism of their influence on the effectiveness of therapy is unclear. CONCLUSION: In CHC, ABs to the C-terminal epitope of NS4a are produced more frequently than those to the median epitope. The presence of ABs to the C-terminal epitope is a predictive marker of a high probability of achieving SVR, regardless of the type of therapy and antibody titer.

[Low concentrations of hepatitis C virus RNA in serologically mild infection.]

Occult HCV infection (OCI) provides significant interest recently. HCV RNA in this case can be detected not in plasma, but in blood cells and/or in liver tissue. In case of antibody genesis impairment anti-HCV detection may lead to negative or "uncertain" result. The aim of the study was to estimate infection type in blood donors and patients with hematological diseases by exploration of samples with uncertain anti-HCV detection results. Blood samples of 30 180 potential blood donors' and 4322 patients with hematological diseases were tested. Comparative analysis of wide pattern of HCV markers was performed. 33 blood donors and 42 patients were enrolled in follow-up examination. Uncertain results of Anti-HCV detection in donors' samples were in 0.18% of cases. Follow-up examination of 33 donors provided discordant results using immunochemiluminescence assay and ELISA. 15.2% donors' samples contained HCV RNA in low concentration. Follow-up observation of 42 patients with incomplete antiviral antibody pattern showed HCV RNA presence in 40.5% cases (21.4% high viremia and 19.0% low viremia). Samples with low RNA concentration contained low titers of anti-core antibodies. Samples with high titers of anti-core antibodies contained high HCV RNA level. Uncertain results of anti-HCV in 15.2% of potential blood donors' samples were confirmed by detection of HCV RNA in low concentration. It proved OCI presence in these individuals and called for testing for wide pattern of HCV markers in addition to routine screening. Patients with hematological diseases showed low level of HCV RNA along with low titers of antibodies against one or two viral antigens.

IMMUNOGENIC PROPERTIES OF RECOMBINANT MOZAIC PROTEINS BASED ON ANTIGENS NS4A AND NS4B OF HEPATITIS C VIRUS.

The aim of the study was to investigate immunogenic properties of mosaic recombinant proteins constructed on the data of hepatitis C virus NS4A and NS4B antigens. Four mosaic recombinant proteins, containing the T and B epitopes of the NS4A and NS4B antigens, were created by genetic engineering methods in the E. coli system. To enhance the immune response they were linked in different variations to the nucleotide sequences of murine interleukin-2 (IL-2), the Neisseria meningiditis lipopeptide, and the T helper epitope of the core protein of hepatitis C virus. The immunogenic properties of these recombinant proteins were analyzed by immunoblotting, ELISA and ELISpot using sera from immunized mice and patients infected with hepatitis C virus. Recombinant proteins specifically reacted with the sera of immunized mice and infected patients in immunoblotting. According to the ELISA data, the predominant formation of antibodies to NS4B was observed when mice were immunized with the recombinant proteins containing both antigens. Analysis of gamma-interferon production by T-lymphocytes upon contact with activated dendritic cells showed in ELISpot that the maximum production of this cytokine was detected when adjuvant components were located at the N- and C-ends of the recombinant protein. The highest level of gamma-interferon production during stimulation with this drug was detected in lymphocytes from the bone marrow and lymph nodes. The recombinant protein containing the T and B epitopes of NS4A and NS4B, murine IL-2 and the lipopeptide Neisseria meningiditis had the greatest immunostimulate effect among the four constructions. This recombinant protein formed nanoparticles of 100-120 nm in size.

DIAGNOSTIC CAPACITY OF DETECTION OF SPECIFIC ANTIBODIES TO PANDEMIC INFLUENZA A(H1N1)PDM09 VIRUS.

Serologic studies occupy a significant place in influenza diagnosis. The article presents an analysis of the developed experimental version of ELISA test-systems for the detection of specific antibodies to the virus influenza A(H1N1)pdm09, and their dynamics at different stages of infection as compared with those of the traditional HAI method. The study included 20 paired samples of serum from patients hospitalized at different stages of the disease with etiology associated with the influenza virus A(H1N1)pdm09. Two groups were formed on the basis of HAI data, which showed the presence or absence of significant growth of specific antibodies to the influenza virus A(H1N1)pdm09. The control group consisted of 20 serum samples from individuals without influenza but with chronic hepatitis C. To examine the virus specific antibody two types of ELISA test systems were used. The first system was intended for the detection of IgM to the influenza virus A(H1N1)pdm09; the second was used for revealing specific IgG. The study showed the accuracy and specificity of detectable IgM and IgG to the virus influenza A(H1N1)pdm09. The dynamics of specific IgG titers in 15 of the 20 pairs of sera was reliable. The increase in titers was more pronounced than in the HAI. IgM against influenza virus could be detected up to 10 days, although reliable dynamics of these antibodies was not detected in paired samples. The test system was specific for the determination of both IgG and IgM antibodies to the influenza virus A(H1N1)pdm09 and significantly more sensitive than HAI. Using this ELISA test system, it is possible to monitor the dynamics of IgG to this virus even in the absence of diagnostic increases in antibody titers in HAI.

[Primary progressive multiple sclerosis: current issues of timely diagnosis]. / Pervichno-progressiruiushchii rasseiannyi skleroz: sovremennoe sostoianie problemy svoevremennoi postanovki diagnoza.

This article presents a review of international data on primary progressive multiple sclerosis (PPMS) and an analysis of factors influencing timely diagnosis of PPMS in a number of regions of the Russian Federation.

[Frequency of detection of different hepatitis C virus subtypes in the Moscow region].

There is a high degree of the hepatitis C virus (HCV) infection in Moscow region. The analysis of different HCV subtypes is caused by the necessity of prognostic estimation of predominant subtypes for the near future because new schemes of antiviral therapy for several HCV genotypes are developed. It is established that subtype 1b (52.1%) prevails in Moscow region. The increase of subtype 3a (37.2%) against the decrease in subtype 1b is observed. In patients infected before 1990 HCV subtype 1b is predominant and liver cirrhosis is detected in 46.7% of cases. The intergenotypic recombinant 2k/1b is registered with the frequency of 0.8% and about 2% of cases of mixed HCV subtypes are detected.

[Hepatitis C virus: therapeutic targets and new drugs].

The viral and patient factors affecting the efficacy of therapy were discussed. The modern data on the molecular mechanism of action of main drugs for hepatitis C therapy were presented. New antiviral drugs (protease inhibitors) and pharmacological substances under development targeted against viral polymerase and protein NS5A were considered.

[Decline of antibodies to hepatitis C virus during antiviral therapy].

The goal of this study was to investigate interrelationship between changes in anti-hepatitis C virus antibody and response to antiviral therapy. The comparative quantitative analysis of antibodies to individual structural and nonstructural viral proteins was done during two years in three patient groups: initial responders, non-responders and a reference group (without therapy). All patients in the treated groups exhibited decrease in the analyzed antibodies to HCV proteins, but with different patterns. The first statistically significant difference in the decrease of virus-specific antibody between initial responders and non-responders was observed within the first three months of therapy beginning. Some treated patients demonstrated decrease in antibody levels to HCV proteins after the end of therapy.

[Humoral response to hepatitis C virus antigens in coinfection with human immunodeficiency virus type 1].

A humoral immune response to individual hepatitis C virus (HCV) antigens was studied in 49 patients at the subclinical stage of HIV-1 infection. These patients, as compared with a group comprising 50 patients with chronic hepatitis C, showed statistically significant higher levels of HCV-specific immunoglobulins G to nucleocapsid protein and the antigens NS3, NS4ab, NS5a. The group of patients with coinfection did not differ from those with chronic HCV monoinfection in detection rates and anti-HCV IgM.

[Antibody responses to consensus sequence of hypervariable region 1 from hepatitis C virus protein E2 in relation to the phase of infection].

Three overlapping peptides containing linear B-cell epitopes and corresponding to the consensus sequence of hypervariable region 1 (HVR1) from protein E2 were studied for their capacity to interact with the sera from patients with acute and chronic hepatitis C. Antibodies to these peptides are detectable with the comparable frequency in both acute and chronic hepatitis C viral infection, other than after viral elimination. A relationship of the outcome of acute Infection to the presence or absence of antibodies to the test peptides has not been established. The level of antibodies against protein E2 HVR1 C-terminus is significantly higher in chronic hepatitis C.

[Detection of hepatitis B virus surface antigen with the use of an optical biosensor]. / Opredelenie poverkhnostnogo antigena virusa gepatita B s pomoshch'iu opticheskogo biosensora.

The detection of hepatitis B virus surface antigen (HBsAg) with the use of a model IAsys+ two-channel optical biosensor is based on the registration of interaction between anti-HBs monoclonal antibodies forming the surface layer of the biochip of the biosensor cuvette and blood serum HBsAg. For the first time a two-channel optical biosensor has been used for the detection of HBsAg in blood serum samples. The comparative analysis of the detection of HBsAg by two methods, viz. with the use of an optical biosensor and the enzyme immunoassay, has demonstrated lower sensitivity, but higher specificity of the detection of this antigen by means of a model IAsys+ biosensor with the biochip, prepared in the process of the work. The main advantages of the biosensor detection lie in the registration of interaction in real time without introducing special markers into the molecules under study.

Virus-specific antibody titres in different phases of hepatitis C virus infection.

This study aimed to examine anti hepatitis C virus (HCV) antibody titres, their changes and differences in acute, chronic and past HCV infection and to examine them after IFN-alpha-therapy. Ninety five patients were studied in a cross-sectional investigation and 18 of them were followed long-term. Titres of IgM and IgG antibodies against core, NS3, NS4 (A + B), NS5A proteins were determined by the third generation enzyme immunoassays. Patients with acute hepatitis C developed IgG antibodies against core protein in titres 1/5-1/800 and against individual NS proteins at the same titres. During the first to second month of acute hepatitis C IgG antibody titres to HCV proteins were very low, but they had risen considerably by the fourth to sixth month. Anti-HCV IgM antibodies were found in half the acute hepatitis serum samples, titres were 1/5-1/40. Sixty individuals with chronic hepatitis C showed IgG antibodies against core in titres 1/800-1/40,000 and against individual NS proteins in titres 1/5-1/20,000. Eight patients with chronic hepatitis C had invariable anti-HCV IgG antibodies over 2-3 years. About 81.7% of chronically infected patients had anti-HCV IgM antibodies in titres 1/5-1/160. Patients with resolution of HCV infection showed only anti-core IgG antibodies (titres 1/5-1/200) or no virus-specific antibodies. Individuals with different response to IFN-alpha-therapy showed two distinct patterns of anti-HCV antibody titres. Acute and chronic HCV infection may be distinguished by anti-core titres.

Mapping and characterization of B cell linear epitopes in the conservative regions of hepatitis C virus envelope glycoproteins.

Forty-eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV-infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera obtained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immunodominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP.

[Detection of the hepatitis B virus surface antigen with a biosensor]. / Detektsiia poverkhnostnogo antigena virusa gepatita B s pomoshch'iu opticheskogo biosensora.

A new method for detection of hepatitis B surface antigen (HBsAg) has been developed. It employees IAsys optical biosensor registration kinetics of HBsAg complexes with monoclonal antibodies. Detection of intermolecular interactions is accompanied by changes of the light refraction coefficient in the sensitive layer of the biosensor cuvette. The main advantage of this diagnostic technique consists in rapid registration of these interactions in real time, without any introduction of special labels into analysing molecules. The optical biosensor method was successfully employed for the detection of HBsAg in human blood serum. A comparative study of HBsAg detection by the optical biosensor and by immunoenzyme analysis demonstrated high specificity of HBsAg detection by this new method.

[Antibodies to the structural proteins of the measles virus studied in some chronic diseases]. / Issledovanie antitel k strukturnym belkam virusa kori pri nekotorykh khronicheskikh zabolevaniiakh.

The qualitative and quantitative analysis of antibodies to measles virus (MV) structural proteins (SP) in sera from patients with chronic glomerulonephritis (CGN), chronic active hepatitis (CAH), and liver cirrhosis (LC) was done. The patients were shown to have neutralizing antibody titres (NAT) higher than those in healthy subjects. An analysis of antibodies to SP was carried out by the radioimmunoprecipitation assay. Antibodies were detected to hemagglutinin, nucleoprotein (NP), fusion protein and to matrix protein (M) both in sera from the patients with these chronic diseases, healthy subjects, and patients with active measles. (The two latter groups were selected for comparison). However, some patients with CAH and LC had no antibodies to M protein in spite of very high NAT. The quantitative analysis of MV antibodies to SP was done only for NP because this antibody had the least individual variations. The quantity of anti-NP antibodies was higher in most sera from patients with chronic diseases than in those from healthy subjects, and reached the level of that in patients with active measles. The presence of MV genome in the peripheral blood lymphocytes from patients CAH, CGN, and LC had been shown earlier. So it is assumed that MV persists in lymphoid tissue where the expression of all SP genes is realized.

[Gastroesophageal reflux and bronchial asthma]. / Zheludochno-pishchevodnyi refliuks i bronkhial'naia astma.

Gastroesophageal reflux was established in 12 out of 38 patients with infectious allergic bronchial asthma out of exacerbation. According to intraesophageal pH-metry, it turned out appreciable in half of the cases. Prospective observations conducted for up to 8 years made it possible to recognize bronchial asthma in 9 out of 63 patients having initial reflux without any bronchopulmonary alterations. Diminution of the tone of the inferior sphincter of the esophagus proved by electromanometry should be regarded as the leading mechanism by which gastroesophageal reflux developed in bronchial asthma patients. In patients having gastroesophageal reflux without bronchopulmonary pathology, the tone of the upper sphincter of the esophagus was normal or elevated whereas in bronchial asthma patients with reflux, the tone of the superesophageal sphincter was naturally lowered, causing microaspiration into the bronchi of the gastric contents flown to the esophagus. It is desirable that metoclopramide (cerucal) which increases the initially reduced tone of the esophageal sphincters may be included into a complex of therapeutic measures elaborated for patients with associated bronchial asthma and gastroesophageal reflux.