Vol 9, No 1 (2019)


Neutrophil as a multifunctional relay in immune system

Dolgushin I.I., Mezentseva E.A., Savochkina A.Y., Kuznetsova E.K.


Over the last two or three decades, a concept regarding functional capacities of neutrophilic granulocytes has been remarkably extended owing to new technologies. Neutrophil-related effector potential against infectious agents, including phagocytosis, production of reactive oxygen and nitrogen species, degranulation coupled with the released multiple enzymes and antimicrobial peptides, and the extracellular trap formation have been studied in detail. In particular, it was found that many of the factors used by neutrophils to directly destroy pathogens also exert regulatory effects on other immune cells as well as on neutrophils in an autocrine manner. In addition, activated neutrophils are able to de novo synthesize a range of biologically active molecules. Neutrophil-related immunoregulatory effects on macrophages, dendritic cells, T-lymphocytes and B-lymphocytes may be mediated both via direct intercellular contacts and indirectly through production of cytokines and other biologically active mediators. Ambiguous proactive and suppressive neutrophil-related effects on immune cells evidence about their important role played both in homeostasis and diverse pathologies, particularly in developing malignant tumors. Diverse, even opposing impact exhibited by neutrophilic granulocytes on immune cells and cells in non-lymphoid tissues, point at their functional plasticity and, probably, heterogeneity. Upon that, modality of effects elicited by neutrophils largely depends on surrounding microenvironment while they exit from the circulation. Widely considered as an inf lammatory response inducer, neutrophils are able to simultaneously trigger mechanisms facilitating restriction and resolution of inf lammatory reaction. Using intravital microscopy in animal models, it allowed to discover that neutrophils are able to re-enter circulation after exiting into the extravascular space, thereby challenging a classical concept about a unidirectional neutrophil migration from the blood vessels to body tissue. In addition, evidence that neutrophils can serve as antigen-presenting cells for T cells under certain conditions and are recruited from inf lammatory sites to draining lymph nodes were also obtained. Although a body of the data were obtained in vitro or in animal models, which therefore require to be further examined and verified, it may be unequivocally acknowledged that a neutrophil-related impact is not only limited to innate immunity.

Russian Journal of Infection and Immunity. 2019;9(1):9-38
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Trefoil factors — new markers of gastrointestinal mucosal barrier

Shestopalov A.V., Dvornikov A.S., Borisenko O.V., Tutelyan A.V.


Trefoil factors (TFFs) are peptides containing a cloverleaf-like structure, which are synthesized in diverse organs including various regions of the human brain (presumably involved in nervous system development and differentiation), thyroid gland, mammary gland, uterus, prostate gland, conjunctiva, respiratory tract, salivary glands, and gall bladder, which peaks in diverse gastrointestinal tract mucosa: TFF-1 — in the stomach, TFF-2 — in the stomach and duodenum, and TFF-3 — in the gut. An increasing body of studies indicate not only about a widely distributed trefoil factors in vivo but also points at their important regulatory functions. In particular, TFFs affect cell adhesion by enhancing epithelial cell migration. Recovery of reversibly damaged epithelial structures called restitution is also facilitated due to TFF-related antiapoptotic effect (anoikis resistance). In contrast, TFF-mediated proangiogenic effects can promote tumor angiogenesis, whereas their immunomodulatory effects include an influence on expression of pro-inflammatory and defense factors (including nitric oxide, cytokines, and defensins). Moreover, TFFs interacting with mucin may increase mucus viscosity, thereby protecting the mucosal layers against ulcerogenic agents. However, bronchial asthma maybe aggravated by elevating mucus viscosity in the respiratory tract due to TFF-2. In addition, TFF expression level is associated with pathogenesis of inflammatory diseases in the gastrointestinal tract. It was shown that modality of changes in TFF level might differ depending on anatomical location and severity of lesions. Changing TFF level plays an important role in oncogenesis. For instance, gastric and colorectal cancer is accompanied by upregulated TFF-1 expression. Importantly, TFF amount is considered as a diagnostic predictor due to being associated with carcinogenesis stage, metastasis as well as sensitivity to chemotherapy in gastrointestinal cancer. In addition, a role potentially played by TFFs in other malignancies including retinoblastoma, breast cancer, and thyroid carcinoma has been extensively examined. Thus, an expanding range of experimental and clinical data evidence that trefoil factors maybe considered as a promising marker of gastrointestinal and oncology diseases.

Russian Journal of Infection and Immunity. 2019;9(1):39-46
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Hexon-based scaffold for generation of diagnostic monoclonal antibodies against diverse adenovirus types

Timoshicheva T.A., Zabrodskaya Y.A., Amosova I.V.


Infectious diseases hold one of the most crucial places in healthcare. In Russia, annual prevalence of infectious diseases comprises around 50 million cases, wherein acute respiratory viral infections cover up to 90%. In non-influenza (non-peak) season, adenoviruses, respiratory syncytial virus, parainfluenza viruses etc. represent the main infectious cause of acute respiratory viral infections. Adenovirus-induced infections are characterized by heterogeneous manifestations poising them both as an interesting challenge, yet difficult in clinical diagnostics. Use of rapid, sensitive and specific tests is of high priority for routine clinical laboratory practice. In Russia, differential diagnostics of adenoviral infections includes a widely used ELISA and immunofluorescence analysis based on polyclonal specific sera. Importantly, a pattern and range of specific reactions depend on origin of animal-derived antibodies and their composition. Introduction of monoclonal antibodies specific to certain viral antigenic epitopes ensures high sensitivity and specificity allowing to reach an expected standardization level for such diagnostic products. Adenovirus hexon protein bears genus-specific antigens and relatively conserved amino acid sequence among diverse adenoviruses. Moreover, it is produced at high amount in infected cells and may be purified in a native form. Moreover, it is produced at high amount in infected cells and may be purified in a native form, thereby posing it as a promising antigen for producing monoclonal antibodies able to detect various adenoviruses types. A panel of adenovirus hexon-specific monoclonal antibodies was generated, which were further examined for biological and diagnostics properties. Western blotting data allowed to conclude that all of monoclonal antibodies generated by us were able to bind to adenovirus hexon oligomers. Specific activity of the new monoclonal antibodies against various adenovirus types was examined by ELISA and indirect immunofluorescence assay. In particular, monoclonal antibodies 4B7 and 6B12 were shown display top specific activity measured by ELISA (antibody titers comprised as high as 10–6). Moreover, monoclonal antibody 6B12 exhibited peak hexon-specific activity assessed in indirect immunofluorescence assay (against various adenovirus types), which resulted in prominent granular nuclear fluorescence in cells infected with adenovirus types 3, 4, 6, and 19. Thus, the data obtained evidence that monoclonal antibodies developed 4B7 and 6B12 maybe potentially used for developing high-quality adenovirus-specific diagnostic assays.

Russian Journal of Infection and Immunity. 2019;9(1):47-56
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Differences in the amino acid composition of the antigen epitopes of the VP7 protein of Russian rotaviruses with the G9 genotype and the vaccine strains RotaTeq, Rotavac, and Rotarix

Morozova O.V., Sashina T.A., Epifanova N.V., Novikova N.A.


Introduction. Rotaviruses of group A (RVA) with genotype G9P[8] are a common cause of acute gastroenteritis in children in Russia. In Nizhny Novgorod, the part of G9P[8] among all RVA strains reached 63.1% during 2016–2017 epidemic season. Two live rotavirus vaccines, RotaTeq and Rotarix have been successfully introduced into the national immunization programs worldwide. In addition, the Indian vaccine Rotavac, based on the strain with G9P[8] genotype, is used on a regional level. The parent strains for all mentioned vaccines were isolated more than 30 years ago. There is no data about phylogenetic analysis and comparative analysis of antigenic epitopes of Russian G9P[8] wild-type isolates and vaccine strains. In the present study, for the first time, we provide a comparative phylogenetic analysis and research of the amino acid composition of the B- and T-cell epitopes of the VP7 protein between Russian rotaviruses with the G9 genotype and the vaccine strains in RotaTeq, Rotarix and Rotavac composition. Materials and methods. The nucleotide and amino acid sequences of the VP7 gene of RVA with genotype G9 were studied. The rotaviruses had been previously isolated from children hospitalized with acute gastroenteritis in the infectious hospital in Nizhny Novgorod during 2011–2016. Results. A phylogenetic analysis of the nucleotide sequences of the VP7 gene showed that the Nizhny Novgorod strains belong to the G9-III allele. Based on the amino acid sequences VP7, three B-cell epitopes (7–1a, 7–1b and 7–2) and two T-cell epitopes (16–28 aa and 40–52 aa) were analyzed. The smallest number of substitutions was found in the RotaTeq vaccine registered in Russia: from 0 to 3 aa differences at the epitope. The same (from 0 to 3 aa differences at the epitope) was found between the wild-type strains RVA and the Rotavac vaccine. The largest number of amino acid differences was found between the vaccine strain Rotarix and the Nizhny Novgorod G9 strains (from 3 to 10 aa at the epitope). Conclusion. In the present work, based on nucleotide sequences VP7 gene, we provide phylogenetic and comparative analyses of the amino acid composition of antigenic epitopes of G9 RVA isolated in Russia vs rotavirus strains in vaccines RotaTeq, Rotavac and Rotarix. The accumulation of mutations in antigenic epitopes can help the virus to escape the immune response. Continuous molecular monitoring of wild-type RVA strains is necessary for estimation of the possible impact of vaccines on the genotype diversity of the rotavirus population in the wild and to monitor the emergence of novel antigenic variants.

Russian Journal of Infection and Immunity. 2019;9(1):57-66
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Immunogenicity and protective efficacy of prime-boost immunization in mice vaccinated with live and inactivated influenza A (H5N1) vaccines

Losev I., Petukhova G., Isakova-Sivak I., Rudenko L.


Avian influenza A (H1N1) in humans is characterized by severe clinical manifestation and high mortality. The main drawback of current human H5N1 vaccines is related to low immunogenicity. Prime-boost vaccination is considered as an effective approach to enhance vaccine immunogenicity. The aim of this study was to compare immune response and protective efficacy of diverse prime-boost immunization protocols: 1) prime and boost with live influenza vaccine (LAIV) А/17/Turkey/Turkey/05/133 (H5N2); 2) prime with LAIV А/17/Turkey/Turkey/05/133 (H5N2) followed by boost with inactivated influenza vaccine (IIV) “Orniflu” (H5N1). Both vaccination protocols were found to increase serum antibody level against homologous and heterologous influenza A virus strains. In particular, serum HAI antibodies were significantly elevated solely after LAIV/LAIV vaccination. A more sensitive sandwich ELISA assay revealed that serum virus-specific IgG antibody levels were significantly increased after both vaccination protocols as well as after a single LAIV or IIV vaccination. Both LAIV and IIV boost increased titers of serum IgG specific against unrelated influenza A (H5N1) strains: homologous A/NIBRG-23 (clade 2.2), A/Indonesia (clade 2.1) and, to a lesser extent, against clade 1 virus A/ Vietnam and even against heterologous А/New York (H1N1). Single LAIV vaccination was also able to induce antibody responses against all strains examined, though to a lesser degree as compared with either prime-boost protocols. However, amount of splenic CD8+  Тcells specific to homologous influenza A virus strain was solely observed after LAIV/IIV vaccination. Moreover, both LAIV and IIV boosting effect demonstrated high protection level against lethal challenge with А (H1N1) WT virus and significantly decreased lung viral titer compared to control group. Furthermore, both regimens resulted in lung virus clearance after non-lethal challenge with clade 1, 2.1 or 2.2 influenza А (H5N1). In conclusion, we demonstrated that both LAIV/LAIV and LAIV/IIV regimens were able to induce cross-clade A (H5N1) response and that prime-boost immunization was a promising approach to improve immunogenicity of influenza A (H5N1) virus vaccine.

Russian Journal of Infection and Immunity. 2019;9(1):67-75
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Induction of HCV-specific cell response in vitro by dendritic cells generated with interferon-α

Chernykh E.R., Oleynik E.A., Leplina O.Y., Tikhonova M.A., Tyrinova T.V., Starostina N.M., Ostanin A.A.


The induction of a strong multi-epitope T-cell response against hepatitis C virus (HCV) plays an important role in eliminating the virus, whereas adoptive response deficiency contributes to chronic and rapid progression of HCV-infection. Since dendritic cells (DCs) are capable of priming naive T lymphocytes and induce an effective immune response, the use of DC-based vaccines to enhance the HCV-specific T cell response is considered as a new approach to treatment of chronic hepatitis C (CHC). The ability of DCs generated from monocytes in the presence of interferon-α and loaded with recombinant HCV proteins Core (1–120) and NS3 (1192–1457) to induce an antigen-specific cellular response in healthy donors and patients with CHC was investigated. The immune response was assessed by proliferative activity and Th1 (IFNγ)/Th2 (IL-4, IL-6) production in mononuclear cells (MNC) cultures, and activation of cytotoxic T-lymphocytes in the degranulation test. We demonstrated that the primary antigen-specific response in MNC cultures of seronegative donors was detected better by stimulation of DCs, loaded with both antigens (DCCore /NS3) than when loaded with a single protein. DCCore/NS3  induced the proliferative response and degranulation of CD8+ T cells in MNC cultures of all tested donors, and in 50% (5/10) cases — IFNγ production. Similarly to donor DCs, DCCore/NS3 of patients with CHC induced a proliferative response in most cases (86%) and IFNγ production in 57% cases. At the same time, the activation of cytotoxic T cells in patients was less frequent (patients vs donors 57 and 100%, respectively), which could be partly due to increased spontaneous degranulation of CD8+ T cells in some patients. The obtained data testify the possibility of using vaccines based on interferon-α-induced DCs for the prevention and treatment of chronic HCV infection.

Russian Journal of Infection and Immunity. 2019;9(1):76-86
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In vitro dendritic cell maturation isolated from healthy people and patients with Staphylococcus aureuscaused chronic osteomyelitis

Rubtsova J.P., Aleynik D.Y., Zhivtsov O.P., Mitrofanov V.N.


Here we present the data comparing maturation of peripheral blood mononuclear cell-derived dendritic cells (DCs) isolated from healthy volunteers and Staphylococcus aureus-positive patients with chronic osteomyelitis. Dendritic cells were cultured in a standard maturation cell medium (RPMI-1640,  supplemented with antibiotics, L-glutamine, 15% calf embryonic serum) added with interleukin-4 and granulocyte-macrophage colony-stimulating factor, followed by adding a stimulating factor cocktail containing interleukin-1β, tumor necrosis factor-α, interleukin-6, and prostaglandin E2. Dendritic cell maturation was analyzed by estimating visual characteristics under Zeiss ODSERVER.Z1 inverted microscope using Axiovision Rel.4.8 imaging software as well as light and phase-contrast microscopy at magnification of ×40, ×100, ×200, ×400, ×630. Dendritic cell immunophenotyping was carried out by using a panel of anti-human monoclonal antibodies: anti-CD80 FITC-conjugated, anti-CD86 (B7–2) PE-conjugated, anti-HLA-DR PC7-conjugated (all from Beckman Coulter, USA), anti-CD14 PerCP-Cy5.5-conjugated, anti-CD83 APC-conjugated, anti-CD40 PE-Cy7-conjugated (Becton Dickinson, USA) as well as isotype-matched control antibodies on the FACS Canto II f low cytometer (Becton Dickinson, USA). It was shown that while maturation dendritic cells derived both from patients or volunteers increased in size and underwent dendrite formation. Moreover, expression of CD86, CD83, CD80, and CD40 markers on dendritic cells derived from patients vs. volunteers was lowered. However, DC stimulation resulted in significantly increased percentage of DCs positive for CD83 DCs co-stimulation molecules CD86, CD80, CD40 chronic osteomyelitis. However, such differences found in immature DCs in both groups disappeared upon maturation, so that expression of the key markers on day 10 was maintained at close level. In particular, expression of CD83 differentiation marker and the CD80 co-stimulation molecule on DCs from patients vs. volunteers was increased stronger. Thus, a maturation potential in DCs isolated from patients with Staphylococcus aureus-caused chronic osteomyelitis was not impaired in vitro. The data obtained open up an opportunity to use dendritic cells as a natural adjuvant-substituting component for development of individualized vaccines in treatment and prevention of recurrent chronic osteomyelitis.

Russian Journal of Infection and Immunity. 2019;9(1):87-94
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A role of methicillinresistant Staphylococcus aureus strains and related molecular genetic features in developing purulent-necrotic forms of the diabetic foot syndrome

Khokhlova O.E., Iwao Y., Kamshilova V.V., Teplyakova O.V., Motova A.I., Drobushevskaya A.I., Perianova O.V., Vinnik Y.S., Potkina N.K., Zdzitowiecki D.E., Yamamoto T.


Purulent-necrotic  complications in patients with diabetic foot syndrome is one of the main causes resulting in amputation and disability, or even lethal outcome. Our study was aimed at investigating a role played by MRSA and related molecular genetic features, as well as antibiotic resistance in developing purulent-necrotic forms of the diabetic foot syndrome in Krasnoyarsk, in the 2010–2016 period. A microbiota profile related to purulent-necrotic complications, antibiotic susceptibility, as well as the molecular genetic features of methicillin-resistant Staphylococcus aureus were examined in 240 patients with diabetic foot syndrome. A bacteriological method was used to investigate microbiota profile related to purulent complications. Antibiotic sensitivity was analyzed by disc-diffusion method; staphylococcal antibiotic sensitivity was evaluated by screening, PCR, solid medium serial dilution, in accordance with the CLSI and EUCAST international recommendations. Genotyping and examining molecular genetic features were performed by using PCR, M-PCR, and sequencing. The data were analyzed by using WHONET (WHO) software. Significance level was set at p <0.05. It was found that microbiota profile linked to purulent-necrotic forms of the diabetic foot syndrome was presented by various Gram-negative microorganisms including Enterobacteriaceae spp. and non-fermenting bacteria accounting for 34.4% and 19.1%, respectively, as well as Gram-positive microorganisms found in 46.5% cases, including S. aureus (18.4% cases). Moreover, microorganisms were characterized by multiresistance to diverse antimicrobial drugs: percent-age of BLDS- and MBL-producers as well as MRSA comprised 36.4%, 30.3%, and 36.4%, respectively. Further, MRSA ST239/spa3(t037)/agr1/SCCmecIII.1.1.2(IIIA)/coaIV/tst+  clone dominated in patients with purulent-necrotic  forms of diabetic foot syndrome admitted to the right-bank and left-bank hospitals in the city of Krasnoyarsk that was characterized by a high virulence level and multidrug resistance. Next, subdominant MRSA genetic variants were presented by ST8/spa1(t008)/agr1/SCCmecIV.3.1.1/CoaIII and ST12/spanew(t156)/agr1/SCCmecUT/coaIorVII characterized by resistance to 1–2 groups of antimicrobials apart from β-lactams. Importantly, similar MRSA genetic variants earlier isolated in patients with other nosological entities common in this geographic region were also mainly verified in patients with purulent-necrotic forms of diabetic foot syndrome admitted to Krasnoyarsk hospitals. In particular, it was demonstrated that MRSA isolates predominantly belonging to the same genetic variants were detected in patients with purulent-necrotic forms of diabetic foot syndrome hospitalized both at the right-bank and left-bank hospitals of the city of Krasnoyarsk. Thus, long-term hospitalization of patients with purulent-necrotic forms of diabetic foot syndrome as well as subsequent repeated admittance to other hospitals in the city of Krasnoyarsk facilitates inter-hospital bacterial strain transmission.

Russian Journal of Infection and Immunity. 2019;9(1):95-106
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Flow cytometry for the analysis of virusneytralizing activity of antirabies serum and immunoglobulin drug

Generalov S.V., Kravtsov A.L., Kozhevnikov V.A., Gavrilova Y.K., Abramova E.G., Nikiforov A.K.


Here, we discuss development of f low cytometry technique for determining level of rabies antibodies in immune sera and immunoglobulin preparations, which is based on measuring fluorescence level in Vero cell line added with a mix of serially diluted anti-rabies serum or immunoglobulin preparation together with Moscow 3253 attenuated rabies virus strain, adapted for reproduction in cell lines. For this, rabies sera and immunoglobulin preparation were diluted 1:20 and 1:200 with PBS, respectively, whereas rabies virus was recommended for use at dose of 0.1 ID50/cell. Cell membrane fixation and permeabilization were performed by using a Cytoperm/Cytofix reagent (BD Pharmingen, USA) containing formaldehyde able to inactivate rabies virus, thus complying with biological safety regulations. Anti-rabies FITC-conjugated immunoglobulin (FGBI ARRIAH, Russia) was recommended for staining, that does not limit using similar reagents. Then, a f low cytometer equipped with a 20 mW argon laser (488 nm emission wavelength, throughput — 500 cells per second) was used for analysis. Vero cells displaying a fluorescence intensity exceeding 10 arbitrary units were considered infected. Flow cytometry allows to precisely measure amount of experimental infected cells as well as degree of infection by evaluating cell f luorescence intensity. Amount of antibodies in the samples examined by us was calculated by building a calibration curve based on depicting data for a standard rabies immunoglobulin with activity of 30 IU/ml (NIBSC, Potters Bar, United Kingdom). A high correlation between the data obtained by us and results from other detection methods used to assess activity of anti-rabies preparations such as biological neutralization of rabies virus in white mice (0.92) and modified FAVN test (0.98) was demonstrated. The comparison of the results of the analysis of the level of rabies antibodies obtained using f low cytometry, and the results obtained in tests in vivo, was carried out for the first time. Moreover, it is worth noting that for the first time the level of anti-rabies antibodies assessed by f low cytometry and in vivo tests was compared. An opportunity to perform rapid and easy-to-do analysis on assessing amount of infected cells by measuring fluorescence intensity in f low cytometry assay would allow to apply this approach for quality control while developing and manufacturing immunobiological anti-rabies preparations.

Russian Journal of Infection and Immunity. 2019;9(1):107-114
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Peculiarities of the phenotype of T-lymphocytes in the dynamics of the postoperating period in patients with peritonite depending on the outcome of disease

Savchenko A.A., Borisov A.G., Cherdancev D.V., Pervova O.V., Kudryavtsev I.V., Belenjuk V.D.


The aim of the study was to investigate the phenotype of blood T-lymphocytes in the dynamics of the postoperative period in patients with widespread purulent peritonitis (WPP) depending on the outcome of the disease. A total of 36 patients aged 30–65 with acute surgical diseases and abdominal injuries complicated by WPP years were examined. Blood sampling was performed before the operation (preoperative period), and also on the 7th, 14th and 21st day of the postoperative period. 40 relatively healthy people of the same age range were examined as a control. Study of the phenotype of blood T-lymphocytes was carried out by the method of 5-color f low cytometry using direct immunof luorescence of whole peripheral blood. Mean levels of fluorescence were used to evaluate the expression levels of surface receptors. It was established that in the preoperative period in patients with WPP regardless of the outcome of the disease against the background of a decrease in the absolute amount of total lymphocytes in the blood the content of T cells increases. The T-lymphocytes of patients with WPP are significantly more intense expressing the CD28 and CD62L receptors than the cells of healthy people. In the preoperative period and within two weeks of postoperative treatment with a favorable outcome of the WPP an increased amount of CD62L+T-lymphocytes is detected in comparison with the indices of patients with an unfavorable outcome of the disease. Other features of the phenotypic composition of T-lymphocytes in patients with a favorable outcome of WPP is an increase in T-regulatory activity which manifests itself both in the preoperative period and within two weeks of postoperative treatment. With a favorable outcome of WPP an increase in the number of activated cytotoxic T-lymphocytes is observed on the 14th day of treatment which with an unfavorable outcome is observed only on the 21st day of treatment. It is assumed that T-cell suppression and activation of cytotoxic T cells are the factors determining a decrease in the intensity of inflammatory processes in WPP and thereby increasing the level of the favorable outcome of the disease. In case of an unfavorable outcome of the disease, postoperative therapy has a weaker or delayed effect on the dynamics of the studied parameters than with a favorable outcome. With a favorable outcome of WPP an increase in the CD3+CD57+cell count is observed already from the second week of treatment and is more pronounced whereas in the case of an adverse outcome of WPP an increase in the level of these cells is observed only at the third week of treatment. However, the level of expression of CD57 on T-lymphocytes is more pronounced throughout the course of the examination with an unfavorable outcome of WPP.

Russian Journal of Infection and Immunity. 2019;9(1):115-127
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Features of immune status in patients with various clinical forms of oral lichen planus

Chuykin S.V., Akmalova G.M., Mirsayapova I.A., Ron G.I., Chernysheva N.D., Khairullina R.M.


Oral mucosal lesions hold one of the lead places in the structure of dental diseases. Oral lichen planus (OLP) considered as a multifactorial disease is of top priority among dermatoses of oral mucosa and red lip border. Diverse putative concepts behind developing lichen planus pathogenesis are discussed including immunoallergic, viral, genetic and membrane-destructive theories. However, an immune theory is thought to play a crucial role in developing lichen planus. Despite documented induction of immune mechanisms, complex interaction between pathological process and normal defense response as well as stirred interest to it, multiple aspects of immunological conflict in lichen planus remain unclear. Few data describing altered immune parameters depending on clinical picture of lichen planus are currently available that suggested to perform the study aimed at examining immune status in patients with various forms of oral lichen planus. There were enrolled 286 oral lichen planus patients (248 females and 38 males), aged 27–84 years. Based on clinical picture, all OLP patients were divided into 6 groups: a typical type, exudative-congestive, erosive-ulcerous, hyperkeratotic, atypical, bullous type. An immune status in peripheral blood samples was evaluated by analyzing innate defense mechanisms as well as humoral and cellular immunity. Multiple altered immune parameters characterized by impaired phagocytic capture and metabolic activity, disimmunoglobulinemia, altered ratio of major immunocompetent cell types and subsets were documented during the study. Moreover, OLP patients with typical, hyperkeratotic, exudative hyperemic, atypical and erosive ulcerous forms were found to have increased amount of CD4+ helper T cells associated with a self-sustained immune response due to suppressed elimination of pathogenic agents consequently resulting in developing autoimmune process. While analyzing immune status in OLP patients, it allowed to find a relationship between dysphagocytosis signs, impaired humoral and cellular immunity as well as various clinical forms of the disease. Thus, it suggests imbalanced mechanisms responsible for pathogen elimination might play a role in OLP pathogenesis, including infectious agents being involved in its development.

Russian Journal of Infection and Immunity. 2019;9(1):128-134
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Complex analysis of correlation interrelations between indicators of humoral and cellular immunity in persons vaccinated against plague

Klyueva S.N., Bugorkova S.A., Goncharova A.Y., Kravtsov A.L., Kudryavtseva O.M., Sandzhiev D.N., Konusheva S.V., Savchenko S.P., Khasykova B.A., Agapov B.L., Shcherbakova S.A.


At present, the search for biomarkers, which indicate the presence of tense antiplague immunity, is continuing. The aim of the study was to determine the parameters of cellular and humoral immunity in persons vaccinated with the plague vaccine according to epidemiological indications and to characterize the correlation interrelations between them in the dynamics of the antiplague response formation. Blood samples from 114 people living in the territory of the Pre-Caspian sandy natural plague foci were investigated. Using commercial kits, the production of cytokines IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, IFNγ, TNFα (Vector-Best, Russia) IL-17A (eBioscience, Austria), immunoglobulins IgG, IgM, IgA (Vector-Best, Russia) was determined on the enzyme immunoassay analyzer LAZURIT (Dynex Technologies, США). Blood leukocyte immunophenotyping was carried out using f luorochrome-labeled monoclonal antibodies Cyto-Stat CD45-FITC,  CD4-PE, CD8-ECD,  CD3-PC5 (Beckman Coulter, США) on the CyAn ADP DakoCytomation f low cytometer (Denmark). Obtained data analysis revealed a number of differences in the production of detectable cytokines (TNFα,  IL-1β, IL-2, IL-4, IL-6, IL-17) between groups of vaccinated and first-vaccinated people. However, human blood cells, regardless of the multiplicity of the graft, retained their functional reactivity and were able to actively secrete the majority of the analyzed cytokines (8 of 10) in response to stimulation with concanavalin A. Diagnostically significant changes in the concentration of IFNγ, TNFα and IL-4 were detected for the characteristics of a specific antiplague response. For these marker cytokines, the greatest number of correlations have been revealed, both with each other and with changes in the level of other parameters: cytokines, immunoglobulins, T cells. The revealed multiple correlation links between the cytokine and immune status indices indicate the activation of both the cellular and humoral components of the antiplague immune response. The informative value of the complex analysis application of correlation interrelations between the humoral and cellular immunity parameters is shown to assess the level of immunological efficacy (actual vaccination) of vaccination in persons vaccinated against plague.

Russian Journal of Infection and Immunity. 2019;9(1):135-146
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Treatment phasespecific frequency and conditions for developing ТТV and HGV infection in children with new onset acute lymphoblastic leukemia

Poslova L.Y., Alekseev A.B., Sergeeva A.V., Kovalishena O.V., Shkarin V.V., Senagina N.E., Brusnigina N.F., Butina T.Y.


Currently, it is believed that TTV and HGV display multifaceted activities in developing diverse acute and chronic processes (disorders affecting the liver, respiratory tract, hematopoiesis oncology diseases etc.). Transfusion of contaminated blood and its components contribute in transmission of HGV and TTV infections. In connection with this, examining a role for HGV and TTV in etiological structure of pediatric liver damage in acute lymphoblastic leukemia (ALL) as well as their relation to developing toxic liver damage against during ongoing therapy is of special interest. Our study was aimed at assessing hospital-acquired HGV and TTV infection and its potential effect on the incidence rate and liver damage intensity in children with acute lymphoblastic leukemia. A rationale for the current study was previously verified hepatitis of unknown etiology excluding viral hepatitis B and C in such patients. In the study, there were examined 99 patients stayed at the Department of Hematology as well as 286 samples from diverse donor bloodderived products. The data obtained were examined by using epidemiological methods (retrospective and near real-time epidemiological analysis, prospective observation), microbiological monitoring, ELISA, PCR followed by analyzing with variation statistics methods. It was found that 51 children, mainly aged 2–4 years (47.1%), were diagnosed with new onset acute lymphoblastic leukemia during a six-year follow-up study. Treatment phase-specific dynamic investigation demonstrated a progressive increase in frequency of HGV-infected ALL patients from 9.8% (day 15 of therapy) up to 45.1% (maintenance therapy). Moreover, a therapeutic intervention was associated with TTV infection detected in 100% cases in baseline TTV DNA-negative patients 100%, and its rate was significantly increased. Thus, our study allowed to demonstrate that 48% and 77% ALL pediatric patients were infected HGV and TTV, respectively, at initial treatment phase (remission induction), that was paralleled with administering the maximum-dose parenteral therapy, including transfusion therapy. Finally, assessing blood donor-derived preparations allowed to detect HGV RNA and TTV DNA in 6.6±1.46% and 19.3±2.9% cases, respectively.

Russian Journal of Infection and Immunity. 2019;9(1):147-154
pages 147-154 views

Blood parasite infection causing inflammatory reactions and benign formations in human thyroid gland

Terletsky A.V., Akhmerova L.G., Evtushenko E.V.


A retrospective examining of cytology specimens obtained and verified by a fine-needle aspiration biopsy from patients with autoimmune thyroiditis and benign thyroid gland (cyst and goiter) formations allowed to note that in thyroid lobes they coincided in various combinations, thus rising a question about their potential etiological relation. In particular, a hemosporidian (blood parasitic) infection was found while analyzing cytology specimens from patients with autoimmune thyroiditis and benign thyroid gland (cyst and goiter) tumors prestained by Romanowsky-Giemsa dye. An evolution of developing intra-thyrocyte hemosporidia was tracked during a long-term detailed analysis of cytology specimens noted above. A panel of select specimens was stained (re-stained) with Schiff reagent according to the Feulgen method to clarify position of thyrocyte DNA and hemosporidian pathogens. Owing to an absorption approach, Romanovsky-Giemsa method allowed to repeatedly use specimens pre-stained with Schiff reagent according to the Feulgen method, wherein fuchsine was incorporated into DNA molecules after they were hydrolyzed by hydrochloric acid to stain specimens into magenta-lilac color. It allowed to identify a parasitic DNA inside developing hemosporidia most probably at exoerythrocytic stage and some erythrocytes cyst-based medusiform structures. Such technique used to stain specimens from patients with autoimmune thyroiditis allowed to localize the thyrocyte nuclear DNA as well as punctate and diffuse cytoplasmic inclusions of parasitic DNA, including magenta-lilac nuclei of different sizes inside erythrocytes. Thyrocyte nuclear DNA as well as punctate and diffuse hemosporidian DNA were distinguished in nodular goiter. Moreover, hemosporidian DNA was identified in a form of magenta-lilac multi-size nuclei inside erythrocytes. In contrast, unstained hemosporidian protoplasm was revealed as light-colored band around erythrocyte nuclei. The intra-erythrocyte nuclear hemosporidian material of different sizes may evidence about various species and/or pathogen generations. Intra-thyrocyte development of hemosporidian infection in patients with goiter results in marked cytoplasmic hyperplasia and its vacuolization associated with thyrocyte nuclear deformation, vacuolization, decreased size and degradation (with highly probability of mutations and deletions), reaching a pre-neoplastic level.

Russian Journal of Infection and Immunity. 2019;9(1):155-161
pages 155-161 views

Climatic prerequisites for changing activity in the natural Crimean-Congo hemorrhagic fever focus in the South of the Russian Federation

Kulichenko A.N., Prislegina D.A.


Subject of study. Examining an impact of the contemporary climatic changes on the natural CCHF focus as well as CCHF incidence in the South of the European part of the Russian Federation. Materials and methods. Annual Epidemiological Report on CCHF Incidence provided by Departments of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing in the subjects of the Southern and the North-Caucasian Federal Districts, CCHF Epizootologic Monitoring Report on the territory of the Volgograd Region, Rostov Region and Stavropol Region were used in the study. Hydrometeorological data were obtained from the database of the All-Russian Research Institute of Hydrometeorological Information — World Data Center, weather station archives as well as The 2011—2016 National Report on the State of Environment in the Stavropol Region. Results. For the first time, climatic factors affecting each phase of the life cycle of ixodid tick Hyalomma marginatum which is the main CCHF virus vector in the South of the European part of the Russian Federation were identified. A direct correlation between seasonal air temperature fluctuations determining ixodid tick count and CCHF incidence was demonstrated. It was found that the average air temperature and accumulated precipitation collectively affect H. marginatum population (e.g., in Stavropol Region). Peak CCHF incidence was demonstrated to correlate with temperature conditions favorable to developing ixodid ticks. In contrast, air temperature fluctuations were noted to negatively impact in the years preceding decreased CCHF incidence. A relation between CCHF incidence and degree of wetting during preceding spring-and-summer as well as autumn seasons affecting vital activity, metamorphosis in preimaginal stages and count of the next-generation H. marginatum was revealed. Moreover, CCHF virus tended to expand geographic range northwards due to an effects of climatic changes. Virus-carrying Ixodidae found on the territory of the administrative districts of the Volgograd Region adjacent to the Volga Federal District evidence about a risk of CCHF virus spreading outside the Southern Regions of the Russian Federation.

Russian Journal of Infection and Immunity. 2019;9(1):162-172
pages 162-172 views

In vivo virulence of Beijing genotype Mycobacterium tuberculosis

Bespyatykh J.A., Vinogradova Т.I., Manicheva O.A., Zabolotnykh N.V., Dogonadze M.Z., Vitovskaya M.L., Guliaev A.S., Zhuravlev V.Y., Shitikov E.A., Ilina E.N.


Mycobacterium tuberculosis Beijing genotype strains comprise 50–80% in Russian Federation, which are divided into the main B0/W148, CladeA, and CAO clusters based on VNTR and SNP analysis. It should be noted that such phylogenetically highly close MTB strains belong to the modern Beijing family, generally demonstrating high transmissibility, association with drug resistance, and prevalence among patients with severe forms of the disease. However, studies on MTB genetic cluster strain-related virulence are scarce and contradictory. Here, we investigated virulence of diverse Mycobacterium tuberculosis strains belonging to the B0/W148, CladeA and CAO clusters and nonclustered strain NK of the Beijing family as well as laboratory strain H37Rv in C57BL/6 mice. It was found that mice infected with NK and B0/W148 vs. CladeA strains revealed the peak and the lowest mortality, respectively, while assessing survival rate in various groups (20 mice per MTB strain examined). Analyzing experimental data in mice demonstrated that all MTB strains were able to cause typical tuberculosis-related pathogenic signs. In particular, time-dependent evaluation of pathological changes (on 1, 3, 7, 14, 21, 28, 60 and 120 day post infection) in the lungs and spleen revealed significant differences among various strains. Tuberculosis progression was observed in the mice infected with B0/W148 and NK strains, whereas CladeA, CAO and H37Rv strains resulted in stabilized course and less marked organs damage. Moreover, we found that bacterial load after infection with Beijing family clustering strains was lower compared to that of the reference H37Rv strain, except NK strain demonstrating the peak bacterial load among the Beijing family comparable to H37Rv strain at 120 dpi. Thus, it was found that the level of virulence between most virulent B0/W148 cluster strain vs. NK strain was similar. Overall, the data obtained indicate that Beijing genotype strains are characterized by a diverse range of phenotypic virulence in vivo.

Russian Journal of Infection and Immunity. 2019;9(1):173-182
pages 173-182 views

Prevalence and molecular-genetic characteristics of hepatitis B virus in HIV-positive individuals at the Far Eastern Federal District

Bazykina E.A., Turkutyukov V.B., Trotsenko O.E., Kotova V.O., Balakhontseva L.A., Butakova L.V., Varnavskaya L.V., Fomenko V.V., Chechulina M.A., Krapivkin A.I., Zinkovskaya S.V., Budai G.I., Yanovich O.A., Lomakina E.A., Lukina Z.I.


Current study was aimed at investigating prevalence of overt and occult hepatitis B infection in HIV-positive individuals as well as molecular-epidemiological characteristics of the circulating hepatitis B virus (HBV) strains in the Far Eastern Federal District (FEFD). A total number of 297 blood serum/plasma samples obtained from HIV-positive patients residing in the FEFD were enrolled in the study. The first control group included 351 blood serum/plasma samples of general population without indication on HIV and HBV-infection that underwent laboratory check up at the Centers for AIDS Prevention and Control. After evaluating the group of HIV-positive patients 20 HIV-HBV positive samples were selected for further detailed analysis. The second control group included 43 patients with chronic hepatitis B. All groups were age and gender-matched. The research included serological and molecular-genetic (real-time PCR, positive for HBV DNA samples underwent clonal sequencing of PCR-amplified HBV P/S gene) assessment of the biological material followed by a phylogenetic analysis of the HBV sequences. Our research revealed that HIV-positive patients are exposed to a higher risk of HBV infection compared to general population enrolled in the study, which is evident from the prevalence of anti-HBcAg antibodies in the groups examined. HIV-positive vs. first control group was positive for anti-HBcAg antibodies at higher rate (35.02%, CI 95: 29.59–40.45% versus 22.22%, CI 95: 17.87–26.57%, p = 0.0003). Abundance of ongoing HBV-infection markers was also higher in HIV-positive individuals compared to general population (6.73%, CI 95: 3.88–9.58% versus 0.85%, CI 95: 0–1.81%, p = 0.0001, respectively). Should be noted, that HBsAg-negative HBV infection in HIV-positive patient cohort comprised 1.01% (CI 95: 0–2.15%) whereas in general population this index was as low as 0.28% (CI 95: 0–0.84%). Virus hepatitis С and D were revealed at higher rate in HIV-positive individuals compared to patients with HIV-negative chronic HBV infection (p = 5.84 × 10–7  and p = 0.000001 respectively). HCV and HDV prevalence rates comprised 50.0% (CI 95: 27.46–77.46%) and 40.0% (CI 95: 17.97–62.03%) in HIV-positive patients. Similar indices in control group were 4.65% (CI 95: 0–10.94%) and 4.65% (CI 95: 0–10.94%), respectively. The phylogenetic analysis of the six isolated HBV sequences showed that the five samples belonged to genotype D, with dominant subtype D2 (verified in 4 cases). Further, HBV genotype С was detected only in one case. The obtained data indicate a necessity for further in-depth diagnostic examination of viral hepatitis in HIV-positive patients to lower a risk of developing life-threatening complications as well as preventing hepatitis spread in human population.

Russian Journal of Infection and Immunity. 2019;9(1):183-192
pages 183-192 views

Russia-wide epidemiological survey of congenital and nosocomial infections in newborns

Ivanova M.V., Mindlina A.Y., Polibin R.V., Ushanova A.V.


High prevalence and great socio-economic impact of nosocomial infections is documented in obstetric hospitals. Objective: to uncover Russia-wide epidemiological features for congenital and nosocomial infections in newborns. Material and Methods: the data covering 2008–2016 period were retrieved from the Federal Statistical Monitoring Form No. 2 “Report on Infectious and Parasitic Diseases”, via the Unified Interdepartmental Information Statistical System. During 2008–2016, incidence of congenital and nosocomial infections in newborns was analyzed in various constituents of the Russian Federation, which were divided into quartile groups including confidence interval. Results: It was found that the increased/decreased/unchanged incidence of congenital infections in newborns was shown in 30, 31 and 13 subjects, respectively. In case of nosocomial infections in newborns, its incidence was increased/decreased/unchanged in 7, 41 and 26 subjects, respectively. Moreover, a congenital/nosocomial infection ratio in newborns was increased/decreased/ unchanged in 40, 15 and 19 subjects, respectively. Based on the morbidity pattern, 9 major groups might be distinguished. In particular, the most abundant (16 subjects) was the group with increased incidence of congenital infections, decreased incidence of nosocomial infections and increased congenital/nosocomial infection ratio in newborns. However, it is worth mentioning that recording of nosocomial infection cases is incomplete in the vast majority of the subjects of the Russian Federation: as few as seven subjects may be highlighted providing a proper registration of nosocomial and congenital infections in newborns such as: Vologda Region, Trans-Baikal Territory, Nizhny Novgorod Region, Omsk Region, Orenburg Region, Penza Region, and Sverdlovsk Region. Conclusion: to fully assess an objective status on nosocomial and congenital infections in newborns, it is necessary that a hospital epidemiologist investigates every single case; ensure applying a standard approach to determine a case of healthcare-associated infection, congenital infections; official recording of all cases; responsibility of all staff members involved in recording healthcare-associated infection cases; regularly reviewed data by using science-based methods. Key words: congenital infections, nosocomial infections, healthcare associated infections, registration, incidence, mortality.

Russian Journal of Infection and Immunity. 2019;9(1):193-202
pages 193-202 views


Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens

Kuzyutina Y.A., Zakharova I.B., Viktorov D.V.


A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating  Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5α cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.

Russian Journal of Infection and Immunity. 2019;9(1):203-208
pages 203-208 views

A verified multiplexed immunoassay for detecting antibodies against infectious pathogens

Poltavchenko A.G., Nechitaylo O.V., Filatov P.V., Ersh A.V.


Baseline screening  of population groups at increased risk of developing infectious diseases (ID) includes a number of ELISA-based analyses, thereby accounting for high cost laborious and time-consuming examination. Earlier wedeveloped a cheap multiplex assay allowing to quickly perform a complex initial testing. Here, we would like to describe a novel kit designed to simultaneously detect antibodies against the six ID pathogens in blood products (plasma, serum etc.): human immunodeficiency virus, hepatitis B and C viruses, cytomegalovirus, Treponema pallidum and Toxoplasma gondii. The kit is based on multiplex dot-immunoassay over flat protein arrays (immune chips) utilizing colloidal gold conjugates and silver development. It allows carrying out complex analysis at room temperature within 70 minutes and does not require highly-qualified personnel. In this article, the results of laboratory tests of the experimental sample of a multiplex kit are compared with commercial ELISA kits from five Russian manufacturers using an array of 240 blood product samples including 200 clinical serum (plasma) samples from patients with ID and 40 plasma samples from donors. It was shown that the multiplex test analysis has a sensitivity and specificity of at least 95%, and optical signals of the dot-assay correlate well with the optical density values obtained using commercial ELISA kits and allow a semiquantitative evaluation of the content of specific antibodies in the sample. Furthermore, multiplex analysis is quicker and cheaper compared to ELISA and can be carried out in field. The kit for multiplex dot-immunoassay of antibodies can provide a complex approach to the diagnosis of ID, significantly simplify initial testing, make it faster, more efficient and affordable for patients.

Russian Journal of Infection and Immunity. 2019;9(1):209-215
pages 209-215 views


Influenza and acute respiratory viral infections



Russian Journal of Infection and Immunity. 2019;9(1):216
pages 216 views

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