Assessment of rat polyclonal antisera's suitability in hemagglutination inhibition assay for influenza surveillance and antigenic mapping.

This paper presents comparative hemagglutination inhibition (HI) assay data obtained using ferret or rat antisera to analyze influenza viruses. The results indicate that rat antisera can be successfully applied both for identification and for antigenic analysis of human influenza A and B viruses. Data gained with rat antisera were comparable to those obtained with ferret antisera. In-depth statistical analysis, based on Confusion Matrix analysis and Receiver Operating Characteristic (ROC) analysis, confirmed good coincidence between ferret antisera-based and rat antisera-based results. Two-dimensional antigenic mapping, based on HI assays using rat and ferret antisera, supported these findings. Both antisera types yielded identical antigenic attributions for the viruses analyzed, and both permitted visualization of contemporary human influenza virus evolutionary trends.

Review article: nonclinical and clinical pharmacology, pharmacokinetics and pharmacodynamics of etrolizumab, an anti-ß7 integrin therapy for inflammatory bowel disease.

BACKGROUND: Novel treatments with superior benefit-risk profiles are needed to improve the long-term prognosis of patients with inflammatory bowel disease (IBD). Etrolizumab-a monoclonal antibody that specifically targets ß7 integrins-is currently under phase III clinical evaluation in IBD. AIM: This review summarises the available pharmacological and pharmacokinetic/pharmacodynamic data for etrolizumab to provide a comprehensive understanding of its mechanism of action (MOA) and pharmacological effects. METHODS: Published and internal unpublished data from nonclinical and clinical studies with etrolizumab are reviewed. RESULTS: Etrolizumab exerts its effect via a unique dual MOA that inhibits both leucocyte trafficking to the intestinal mucosa and retention within the intestinal epithelial layer. The gut-selectivity of etrolizumab results from its specific targeting of the ß7 subunit of α4ß7 and αEß7 integrins. Etrolizumab does not bind to α4ß1 integrin, which mediates lymphocyte trafficking to tissues including the central nervous system, a characteristic underlying its favourable safety with regard to progressive multifocal leucoencephalopathy. Phase I/II studies in patients with ulcerative colitis (UC) showed linear pharmacokinetics when etrolizumab was administered subcutaneously at 100 mg or higher once every 4 weeks. This dose was sufficient to enable full ß7 receptor occupancy in both blood and intestinal tissues of patients with moderate to severe UC. The phase II study results also suggested that patients with elevated intestinal expression of αE integrin may have an increased likelihood of clinical remission in response to etrolizumab treatment. CONCLUSION: Etrolizumab is a gut-selective, anti-ß7 integrin monoclonal antibody that may have therapeutic potential for the treatment of IBD.

PROBLEMS OF ISOLATION, IDENTIFICATION AND ANTIGENIC CHARACTERIZATION OF RECENT HUMAN A(H3N2) INFLUENZA VIRUSES.

Human A (H3N2) influenza viruses are distinguished by a high rate of evolution and regularly cause epidemics around the world. Their ability to adapt and to escape from the host's immune response and to change their receptor specificity is very high. Over the past 20 years, these viruses have lost the ability to agglutinate red blood cells of chickens and turkeys and have practically ceased to propagate in chicken embryos - the main source of influenza vaccines. Isolation of viruses in the MDCK cell culture led to the selection of strains that lose one of the potential glycosylation sites. Many of the A (H3N2) strains have acquired mutations in neuraminidase, which distort the results of antigenic analysis in the hemagglutination inhibition test - the cornerstone method for the analysis of the match between viral isolates circulating in human population to strains selected for the influenza vaccines. In this regard, the characteristics of the antigenic properties of influenza A (H3N2) viruses by traditional methods become poorly informative, and the selection of vaccine strains of this subtype is erroneous, which is reflected in the discrepancy between vaccine and circulating A (H3N2) viruses in recent years (2013-2014, 2014 -2015, 2015-2016). The search, development and implementation of new algorithms for the isolation and antigen analysis of influenza A (H3N2) viruses are extremely urgent.

[IMPACT OF VARIOUS MULTIPLICITY OF INFECTION OF INFLUENZA A VIRUS ON PROLIFERATION AND APOPTOSIS INDUCTION IN CULTURED CELL LINES OF LYMPHOCYTIC AND MONOCYTIC ORIGIN (JURKAT, NC-37, THP-1 AND U-937)].

The severity of disease caused by influenza A infection depends not only on biological characteristics of the virus but also on the number of viral particles than penetrate the body. T- and B-lymphocytes as well as monocytes (macrophages) play a key role in the development of cell-based and humoral immunity as well as influenza virus elimination from the body. The present study describes the effect of influenza A virus infection on cell proliferation and induction of apoptosis in human cultured cell lines of T-, B-lymphocytic and monocytic origin infected with various multiplicity of infection (moi). Low moi of the virus stimulated cell proliferation; maximal effect has been registered 3-4 days after infection. But the fate of T-cells, B-cells and monocytes after initial infection was different: Jurkat cells continued intense proliferation while proliferation of NC-37, THP-1 and U-937 cells lowered. Prolonged (for 3 passages) cultivation of Jurkat, NC-37 and U-937 cell lines has shown that infection of these cell lines not only with low but also with medium and high moi also leads to stimulation of proliferation. Using a variety of methods for the detection of viral reproduction has clearly shown that infection of non-permissive human T-, B-cells and monocytes with influenza A virus leads to latent infection. So, low moi interferes with normal formation of viral particles, which in turn might stimulate cell proliferation and then be followed by induction of apoptosis. Antiviral drags rimantadine and ribavirin suppressed virus-induced cell proliferation; at the same time, induction of apoptosis was suppressed only by rimantadine and was enhanced by ribavirin. The data obtained provide strong support for the role of influenza A virus in the observed effects.

[THE COMPARATIVE ANALYSIS OF EFFECTIVENESS OF QUICK TESTS IN DIAGNOSTIC OF INFLUENZA AND RESPIRATORY SYNCYTIAL VIRAL INFECTION IN CHILDREN].

The analysis was implemented concerning diagnostic parameters of commercial quick tests (immune chromatographic tests BinaxNOW Influenza A&B and BinaxNow RSV Alere, Scarborough Inc., USA) under detection of antigens of influenza virus A and respiratory syncytial virus in clinical materials. The polymerase chain reaction in real-time and isolation ofviruses in cell cultures. The analysis of naso-pharyngeal smears from 116 children demonstrated that sensitivity and specifcity of detection of influenza virus A using device mariPOC in comparison with polymerase chain reaction made up to 93.8% and 99.0% correspondingly at total concordance of results of both techniques as 98.3%. At diagnosing of respiratory syncytial virus using device mariPOC parameters made up to 77.3%, 98.9% and 862% as compared with polymerase chain reaction. The sensitivity, specificity and total concordance of results of immune chromatographic tests BinaxNOW in comparison ofpolymerase chain reaction made up to 86.7%, 100% and 96.2% correspondingly at detection of influenza virus A and 80.9%, 97.4% and 91.6% correspondingly at detection of respiratory syncytial virus. In comparison with isolation technique in cell cultures sensitivity of system mariPOC and immune chromatographic tests proved to be in 1.3-1.4 times higher at detection of influenza virus A and in 1.7-2 times higher in case of isolation of respiratory syncytial virus. There is no statistically significant differences between diagnostic parameters received for mariPOC and immune chromatographic tests at diagnosing influenza virus A and respiratory syncytial viral infection.

[Comparative study of MDCK and CaCo-2 cell lines for influenza virus isolation].

Study of effectiveness of CaCo-2 cell line for influenza virus isolation was carried out. It was shown that the properties of this cell line strongly depended on the source of its origin and cultivation conditions. The infectious activity of the influenza viruses on CaCo-2 cell line was virtually the same as in the MDCK cell line. The rate of the viral isolation was virtually identical for both cell lines tested, but viruses from post-mortem materials were isolated only in CaCo-2 cell line. In general, the CaCo-2 line is believed to be a valuable cell line for virological research, particularly for influenza virus isolation.

[Role of cellular cytoskeleton in influenza A infectious cycle].

Influenza remains a significant social threat especially regarding the emergence of new mutant or reassortant strains. Measures of prophylaxis do not provide complete and stable protection from infection and the use of antivirals results in high-level occurrence of resistant forms of viruses. Nowadays more and more attention is paid to find new targets for antiviral therapy that are not directly connected with virus proteins but can act indirectly through cellular mechanisms involved in viral replication. This approach requires complete understanding of various cellular pathways used by influenza virus. Here we present a brief overview of interactions between influenza A virus and the cell cytoskeleton. This interaction is initiated from the very beginning of influenza infection--adsorption--and continues with endocytosis, release of viral RNP and its entry into the nucleus. The role of cytoskeleton during the late stages of infection is also of great importance. It takes part in NP translocation from the nucleus to the cytoplasm, virus assembly and budding. The presence of cellular actin in certain influenza virions is therefore not accidental but reflects the peculiarities of interaction between a virus and a host cell.

[Evolutionary variability of influenza B viruses in Russian Federation in 2005-2012].

Specific traits of influenza B viruses circulation in Russia and worldwide in 2005-2012 were studied and the amount of influenza B viruses in the whole population of influenza viruses isolated in Russia was estimated. The trend toward antigenic drift for both Victoria and Yamagata lineages was characterized. The genetic analysis revealed amino acid changes that influenced the antigenic properties of the viruses. The match of the epidemic isolates and vaccine strains was corroborated.

[Development of influenza surveillance in Russia in the system of the WHO national influenza center].

Analysis of development influenza activity season 2010-2011 is presented. Significant participation of influenza A(H1N1)pdm09 virus and influenza B of Victoria lineage virus in the epidemic morbidity structure with minor participation ofA(H3N2) virus was revealed. The influenza viruses isolated in Russia according to antigenic properties were similar to the strains included in the vaccine composition. Drift variants of influenza A(H1N1)pdm09 viruses isolated in Astrakhan and St.-Petersburg were recognized using WHO CC in London as representatives of three new genetic groups.

[Analysis of pandemic influenza in Russia as a part of the global process based on data of an influenza monitoring reference center].

AIM: Characterization of features of influenza pandemic development in Russia in relation to global process. MATERIALS AND METHODS: Pandemic monitoring was performed by using results of integrative analysis of laboratory diagnostic and population morbidity data from 49 supporting bases of Federal center of influenza from various cities in Russian Federation. Isolation of influenza virus was carried out in MDCK cells and chicken embryos under BSL-3 conditions. Reference virus A/California/07/09 obtained from CDC (Atlanta, USA) and antisera against this strain contained in WHO kit were used for antigenic analysis; rat antisera, new monoclonal antibodies against pandemic influenza virus developed by Research institute of influenza were also used. RESULTS: Based on PCR monitoring during epidemic peak, rate of pandemic influenza identification reached 45-49% of examined patients. About 53% of lethal cases of respiratory infections were caused by pandemic influenza virus, while predominately young people died from pneumonia and acute respiratory distress syndrome. Russian isolates generally were antigenically and genetically similar to the parent pandemic strain--influenza virusA/California/07/09, but contained S203T substitution in hemagglutinin. A number of strains contained D222G mutation that is responsible for the expansion of substrate specificity, as well as strain specific substitutions in hemagglutinin and neuraminidase molecules. The investigated isolates were resistant to remantadin, but sensitive to oseltamivir. CONCLUSION: Due to the formation of population immunity after the end of the first pandemic wave new drift variants of the virus capable of overcoming this formed immunity should be expected that apparently will require the correction of vaccine composition for the 2011 - 2012 season.

[Pandemic influenza 2009 in Russia. Characteristics of the isolation and biological properties of viruses].

Research Institute of Influenza, Ministry of Health and Social Development of Russia, Saint Petersburg The characteristics of the isolation of pandemic influenza A(H1N1)v viruses were studied on chick embryos (CE) and MDCK cell culture. The materials (nasal swabs and autopsies) were collected in different regions Russia in the period from 20 July to 30 December 2009. The paper gives the data of the antigenic analysis of isolates, their capacity to multiply in different species-specific and tissue cell cultures. The viruses isolated on CE were shown to have higher hemagglutination titers and to be more stable. Isolation from the autopsies was effective only on CE. All the test cell lines other than MDCK were insensitive to the isolated pandemic influenza strains. The antigenic analysis showed no significant antigenic drift of the viruses isolated during the first wave of the pandemic in the Russian Federation.

[The 2009 pandemic influenza in Russia. I. Diagnosis and molecular biological characteristics of the virus].

The analysis of 1558 clinical samples revealed influenza virus A(H1N1v) RNA in 339 patients with influenza and 163 fatal cases,which was made in May to December 2009. Data on the antigenic properties of more than 250 of pandemic virus strains isolated at the Research Institute of Influenza and the molecular genetic characteristics of 31 strains are presented. All the test isolates were found to have the S203 substitution in hemagglutinin, which was characteristic of one of 5 minor genome A(H1N1v) virus variants found in the United States and Mexico in 2009. All the test strains contain the S31N substitution in the M2 protein, which determines viral resistance to adamantine, and have no H275Y substitution in neuraminidase, which determines oseltamivir resistance. The substitution of amino acid residue of Asp to Gly at position 222 of HA was found in 8 (73%) of 11 isolates from postmortem lung and trachea samples and in 2 (10%) of 20 isolates from nasopharyngeal swabs. The determination of the pathogenic role of this substitution calls for further investigations.

A humanized monoclonal antibody targeting the ß7 integrin selectively blocks intestinal homing of T lymphocytes.

BACKGROUND AND PURPOSE: rhuMAb Beta7 is a humanized anti-human ß7 monoclonal antibody currently in phase I in inflammatory bowel disease. rhuMAb Beta7 binds the ß7 subunit of the integrins α4ß7 and αEß7, blocking interaction with their ligands. These integrins play key roles in immune cell homing to and retention in mucosal sites, and are associated with chronic inflammatory diseases of the gastrointestinal tract. The goal of this study was to evaluate the mucosal specificity of rhuMAb Beta7. EXPERIMENTAL APPROACH: We assessed the effect of murine anti-Beta7 on lymphocyte homing in mouse models of autoimmune disease. We also compared the effect of rhuMAb Beta7 on circulating mucosal-homing versus peripheral-homing T cells in naïve non-human primates. KEY RESULTS: In cynomolgus monkeys, occupancy of ß7 integrin receptors by rhuMAb Beta7 correlated with an increase in circulating ß7(+) mucosal-homing lymphocytes, with no apparent effect on levels of circulating ß7(-) peripheral-homing lymphocytes. rhuMAb Beta7 also inhibited lymphocyte homing to the inflamed colons of severe combined immunodeficient mice in CD45RB(high) CD4(+) T-cell transfer models. Consistent with a lack of effect on peripheral homing, in a mouse model of experimental autoimmune encephalomyelitis, anti-ß7 treatment resulted in no amelioration of CNS inflammation. CONCLUSIONS AND IMPLICATIONS: The results presented here suggest that rhuMAb Beta7 selectively blocks lymphocyte homing to the gastrointestinal tract without affecting lymphocyte trafficking to non-mucosal tissues. rhuMAb Beta7 provides a targeted therapeutic approach with the potential for a more attractive benefit:risk ratio than currently available inflammatory bowel disease therapies.

[Influence of rimantadine, ribavirine and triazavirine on influenza A virus replication in human monolayer and lymphoblastoid cell lines].

The influence of antivirals, such as rimantadine, ribavirine and triazavirine on influenza virus replication in human cell cultures was evaluated. All the antivirals inhibited viral nucleoprotein NP synthesis. The strongest effect was shown for ribavirine in lung carcinoma A-549 cells and endothelial ECV-304 cells. Hoechst-33258 staining revealed induction of apoptosis in all the cell lines. Rimantadine and ribavirine inhibited virus-induced apoptosis while ribavirine enhanced it. The effect was registered in monolayer cell cultures as well as in suspension cell cultures. The influence of the antiviral drugs on the virus-induced cell proliferation in the suspension cell cultures is also described.

[Comparative study of the differential susceptibility of different cell lines to pandemic H1N1v influenza viruses and avian influenza, swine influenza, and human influenza viruses].

The proliferation characteristics of influenza viruses of different origin were tested in various human and animal cell cultures. Pandemic H1N1v influenza and swine influenza viruses were shown to have a low infectious activity in virtually all the test lines. In spite of this, the replication of this group of viruses may be detected by de novo NP synthesis. These viruses are able to activate programmed cell death. Moreover, a low inoculative virus dose exerts a stimulating effect on cell proliferation in both suspension and monolayer cell lines.

[Etiological characteristics of the influenza epidemics of 2006-2009 in the Russian Federation (according to the data of the Research Institute of Influenza, North-Western Branch, Russian Academy of Medical Sciences)].

The basic trends in the evolution of influenza A and B in the Russian Federation during the epidemic seasons of 2006-2009 were studied on the basis of an antigenic analysis of 1774 Influenza isolated at the Research Institute of Influenza (RII), North-Western Branch, Russian Academy of Medical Sciences, and sent from resting bases (the regional centers of the Russian Inspectorate for the Protection of Consumer Rights and Human Welfare, which collaborate with the RII). Although the trends in the substitution of representative strains generally coincide with the world patterns, the authors revealed some specific features of the antigenic drift of influenza viruses in the Russian Federation and regional varieties. Data on some biological properties and those of the antigenic analysis of the first pandemic influenza A(H1NI)v strains isolated at the RII from Saint Petersburg patients in July-August 2009 are also given in the paper.

[Study of the antiviral activity of Russian anti-influenza agents in cell culture and animal models].

The study of the antiviral activity of Russian anti-influenza agents in the cultured MDCK cells demonstrated that arbidol and ribavirin inhibited the reproduction of various influenza A virus strains, including rimantadine- and ozeltamivir-resistant variants, as well as influenza B viruses (IC50 2-8.5 microg/ml). Rimantadine at concentrations of 1-5 microg/ml completely inhibited the reproduction of reference and ozeltamivir-resistant influenza A virus strains, and it had no effect on the reproduction of influenza B viruses and rimantadine-resistant influenza A viruses. Arbidol and ribavirin also inhibited the reproduction of pandemic influenza A/California/04/2009(H1N1), A/California/07/2009(H1N1), and A/Moscow/01/2009(H1N1)swl viruses in the cultured MDCK cells (IC50 = 1.5-4.0 microg/ml) while rimantadine had no effect on their reproduction. The cultured cells showed no significant antiviral activity of ingavirin at nontoxic concentrations (up to 200 microg/ml) against all study strains of influenza A and B viruses, including pandemic A(H1N1) influenza virus strains. The activity of rimantadine, arbidol, and ingavirin was found on a model of Influenza pneumonia in mice infected with their adopted influenza A/Aichi/2/69(H3N2) virus. The preventive efficacy of the three test agents was similar and most pronounced when they were used 96 hours before infection, by preventing 40-50% death in the animals and their body weight loss and by increasing their survival by 1.3-1.5 times. Arbidol and rimantadine were more effective when used for treatment and prophylaxis in doses of 30 and 10 mg/kg/day, respectively, by protecting the infected animals from 60-80% death, increasing their survival by 1.7-2 times, and preventing their body weight loss as compared with the control. The same experiments with ingavirin showed that this agent was less effective than arbidol and rimantadine. Thus, arbidol and rimantadine have a pronounced antiviral infection in both cell culture and a model of influenza pneumonia. The found efficacy of ingavirin on an integral model of murine influenza pneumonia without its activity in the cultured cells is likely to be due to other pharmacological properties of the drug rather than its direct virus-specific action.

Review paper: preclinical models of psoriasis.

Psoriasis is the most common autoimmune disease in man and is characterized by focal to coalescing raised cutaneous plaques with consistent scaling and variable erythema. The specific pathogenesis of psoriasis is not completely understood, but the underlying mechanisms involve a complex interplay between epidermal keratinocytes, T lymphocytes as well as other leukocytes (including dendritic cells and other antigen presenting cells [APCs]), and vascular endothelium. Mirroring the complexity of mechanisms that underlie psoriasis, there are a relatively large number of models of psoriasis. Each model is based on a slightly different pathogenic mechanism, and each has its similarities to psoriasis as well as its limitations. In general, psoriasis models can be very broadly divided on the basis of the pathogenic mechanisms that interplay to cause psoriasis, with the addition of several relatively poorly defined spontaneous murine mutant models. Other than the spontaneous mutant models, murine models of psoriasis can be divided into those that are genetically engineered (transgenic and knockout-with manipulation of either the epidermis, leukocytes, or the endothelium), and those that are induced (either by immune transfer or by xenotransplantation of skin from psoriatic patients). In addition to the murine models, in vitro human epidermal models have recently become more widely utilized. While no one single model of psoriasis is ideal, many have proven to be extremely valuable in investigating and better understanding the molecular mechanisms that underlie the complex interplay between epidermal keratinocytes, the innate and adaptive immune system, and the vascular endothelium in psoriasis.

CD4+CD45RBHi T cell transfer induced colitis in mice is accompanied by osteopenia which is treatable with recombinant human osteoprotegerin.

BACKGROUND AND AIMS: Transfer of CD4+CD45RBHi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. METHODS: CD4+CD45RBHi or CD4+CD45RBLo T cells (4 x 10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. RESULTS: Mice that received CD4+CD45RBHi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4+CD45RBLo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4+CD45RBHi or CD4+CD45RBLo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4+CD45RBHi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4+CD45RBHi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG. CONCLUSIONS: CD4+CD45RBHi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.