Nina V. Tikunova

Combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and EBV antigen

Multiple sclerosis (MS) is a widespread neurodegenerative autoimmune disease with unknown etiology. It is increasingly evident that, together with pathogenic T cells, autoreactive B cells are among the major players in MS development. The analysis of myelin neuroantigen‐specific antibody repertoires and their possible cross‐reactivity against environmental antigens, including viral proteins, could shed light on the mechanism of MS induction and progression. A phage display library of single‐chain variable fragments (scFvs) was constructed from blood lymphocytes of patienst with MS as a potential source of representative MS autoantibodies. Structural alignment of 13 clones selected toward myelin basic protein (MBP), one of the major myelin antigens, showed high homology within variable regions with cerebrospinal fluid MS‐associated antibodies as well as with antibodies toward Epstein‐Barr latent membrane protein 1 (LMP1). Three scFv clones showed pronounced specificity to MBP fragments 65–92 and 130–156, similar to the serum MS antibodies. One of these clones, designated E2, in both scFv and full‐size human antibody constructs, was shown to react with both MBP and LMP1 proteins in vitro, suggesting natural cross‐reactivity. Thus, antibodies induced against LMP1 during Epstein‐Barr virus infection might act as inflammatory trigger by reacting with MBP, suggesting molecular mimicry in the mechanism of MS pathogenesis.—Gabibov, A. G., Belogurov, A. A., Jr. Lomakin, Y. A., Zakharova, M. Y., Avakyan, M. E., Dubrovskaya, V. V., Smirnov, I. V., Ivanov, A. S., Molnar, A. A., Gurtsevitch, V. E., Diduk, S. V., Smirnova, K. V., Avalle, B., Sharanova, S. N., Tramontano, A., Friboulet, A., Boyko, A. N., Ponomarenko, N. A., Tikunova, N. V. Combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross‐react with myelin basic protein and EBV antigen. FASEB J. 25, 4211–4221 (2011). www.fasebj.org

Evolutionary time-scale of primate bocaviruses

Human bocavirus (HBoV) is associated with acute gastroenteritis in humans, occurring mostly in young children and elderly people. Four bocavirus genotypes (HBoV1-HBoV4) have been found so far. Since there were no data on the contribution of HBoV to gastroenteritis in Russia, 1781 fecal samples collected from infants hospitalized with acute gastroenteritis in Novosibirsk, Russia during one year were tested for the presence of nucleic acids from HBoV and three major gastrointestinal viruses (rotavirus A, norovirus II, and astrovirus). HBoV was detected only in 1.9% of the samples: HBoV1 was detected in 0.6% and HBoV2, in 1.3%. Complete genome sequencing of three Novosibirsk isolates was performed. An evolutionary analysis of these sequences and the available sequences of human and great apes bocaviruses demonstrated that the current HBoV genotypes diverged comparatively recently, about 60-300years ago. The independent evolution of bocaviruses from chimpanzees and gorillas commenced at the same time period. This suggests that these isolates of great apes bocaviruses belong to separate genotypes within the species of human bocavirus, which is actually the primate bocavirus. The rate of mutation accumulation in the genome of primate bocaviruses has been estimated as approximately 9×10(-4)substitutions/site/year. It has been demonstrated that HBoV1 diverged from the ancestor common with chimpanzee bocavirus approximately 60-80years ago, while HBoV4 separated from great apes bocaviruses about 200-300years ago. The hypothesis postulating independent evolution of HBoV1 and HBoV4 genotypes from primate bocaviruses has been proposed.

Molecular epidemiology of noroviruses associated with sporadic gastroenteritis in children in Novosibirsk, Russia, 2003-2012.

Noroviruses (NoVs) are an important cause of acute gastroenteritis worldwide. To monitor the molecular epidemiology of NoVs genogroup II (GII) in Novosibirsk, Russia, a total of 10,198 stool samples from young children hospitalized with acute gastroenteritis and two asymptomatic comparison groups were collected from 2003 to 2012. All samples were screened for the presence of NoV GII, rotavirus, and astrovirus by RT‐PCR. The prevalence of NoV in gastroenteritis cases was 13.1%, varying from 7.1% to 21.3% in different seasons. Rotavirus and/or astrovirus were detectable in 25% of the NoV‐positive samples. NoV was detected throughout the year with a seasonal increase during winter months. Based on sequence analysis of regions D and/or C within the VP1 gene, 892 identified NoV strains were divided into nine genotypes—GII.3 (51%), GII.4 (44%), GII.6 (2%), as well as GII.1, GII.2, GII.5, GII.7, GII.16, and GII.21 (totally, 3%). The prevalence of NoV in the comparison groups was considerably lower (∼2.5%); only GII.4 (n = 6), GII.21 (n = 2) and GII.1 (n = 1) genotypes were revealed. Based on phylogenetic analysis of the ORF1/ORF2 junction region sequences, GII.P21/GII.3 recombinant and GII.P4/GII.4 were prevalent genotypes (totally, 93%) and their ratio changed every season. The median age of children with NoV infection was 6.6 months (range, <1–35 months), but it was different depending on NoV genotype. Children infected with the NoV GII.3 were younger (median 6.2 months) than GII.4‐positive patients (median 9.1 months). This is the first long‐term systematic study of NoV molecular epidemiology in Russia. J. Med. Virol. 87:740–753, 2015.

Comparative Metagenomic Profiling of Symbiotic Bacterial Communities Associated with Ixodes persulcatus, Ixodes pavlovskyi and Dermacentor reticulatus Ticks

Ixodes persulcatus, Ixodes pavlovskyi, and Dermacentor reticulatus ticks inhabiting Western Siberia are responsible for the transmission of a number of etiological agents that cause human and animal tick-borne diseases. Because these ticks are abundant in the suburbs of large cities, agricultural areas, and popular tourist sites and frequently attack people and livestock, data regarding the microbiomes of these organisms are required. Using metagenomic 16S profiling, we evaluate bacterial communities associated with I. persulcatus, I. pavlovskyi, and D. reticulatus ticks collected from the Novosibirsk region of Russia. A total of 1214 ticks were used for this study. DNA extracted from the ticks was pooled according to tick species and sex. Sequencing of the V3-V5 domains of 16S rRNA genes was performed using the Illumina Miseq platform. The following bacterial genera were prevalent in the examined communities: Acinetobacter (all three tick species), Rickettsia (I. persulcatus and D. reticulatus) and Francisella (D. reticulatus). B. burgdorferi sensu lato and B. miyamotoi sequences were detected in I. persulcatus and I. pavlovskyi but not in D. reticulatus ticks. The pooled samples of all tick species studied contained bacteria from the Anaplasmataceae family, although their occurrence was low. DNA from A. phagocytophilum and Candidatus Neoehrlichia mikurensis was first observed in I. pavlovskyi ticks. Significant inter-species differences in the number of bacterial taxa as well as intra-species diversity related to tick sex were observed. The bacterial communities associated with the I. pavlovskyi ticks displayed a higher biodiversity compared with those of the I. persulcatus and D. reticulatus ticks. Bacterial community structure was also diverse across the studied tick species, as shown by permutational analysis of variance using the Bray-Curtis dissimilarity metric (p = 0.002). Between-sex variation was confirmed by PERMANOVA testing in I. persulcatus (p = 0.042) and I. pavlovskyi (p = 0.042) ticks. Our study indicated that 16S metagenomic profiling could be used for rapid assessment of the occurrence of medically important bacteria in tick populations inhabiting different natural biotopes and therefore the epidemic danger of studied foci.

Heavy–light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation ☆

The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

High evolutionary rate of human astrovirus.

Human astrovirus is one of the etiological agents of acute gastroenteritis in humans, mostly in young children and elderly people. Complete genome sequencing of four human astrovirus strains isolated in Novosibirsk, Russia was performed. Analysis of these sequences and the sequences available in GenBank database has detected numerous potential recombination breakpoints. For the first time the rate of human astrovirus evolution was estimated based on the genome fragments without recombination breakpoints; the determined rate is typical of the RNA viruses with high evolutionary rate, amounting to approximately 3.7 × 10(-3) nucleotide substitutions per site per year, and for the synonymous changes, 2.8 × 10(-3) nucleotide substitutions per site per year.

Recombinant Analogs of a Novel Milk Pro-Apoptotic Peptide, Lactaptin, and Their Effect on Cultured Human Cells

We recently isolated and characterized a human milk peptide, lactaptin, which induced apoptosis of cultured human MCF-7 cells. Lactaptin was identified as a proteolytic fragment of human kappa-casein. Here, we generated two recombinant analogs of the peptide, RL1 and RL2, containing truncated and complete amino acid sequences of lactaptin, respectively. Analogs were produced in E.coli, purified and assayed for biological activity on cultured human MCF-7 cells. RL1 was shown to induce only a small decrease in cell viability, whereas RL2 lowered the viability of MCF-7 cells by 60%. This reduction in MCF-7 cell viability was associated with apoptosis, which was indicated by phosphatidilserine externalization and caspase-7 activation. The viability of A549 and Hep-2 cells was also reduced by RL2, albeit to a lesser degree than seen with MCF-7 cells; this reduced viability was not accompanied by apoptosis. Non-malignant human mesenchymal stem cells (MSC) were completely resistant to RL2 action.

A study of the human bocavirus replicative genome structures.

The complete genomes of two human bocavirus 4 (HBoV4) isolates recovered in 2011 in Novosibirsk, Russia have been determined. A set of primers was designed based on the determined and previously published HBoV sequences; this primer pair was able to detect all possible HBoV replicative intermediates. This primer set was used to assay all HBoV genotypes and detected only those structures that correspond to an episomal form of this viral genome. Also, for the first time, head-to-tail nucleotide sequences have been determined for HBoV4. Secondary structures of the terminal noncoding regions (NCRs) of episomal forms have been computed for all HBoV genotypes, as well as for the canine bocavirus. Conserved secondary structures in episomal NCRs, which are likely to play an important part in the replication of bocaviruses, were found. NCR heterogeneity in the genomes of individual HBoV isolates has been shown for the first time.

Genetic variability of Rickettsia spp. in Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia: Identification of a new Candidatus Rickettsia species

Rickettsia spp. are the causative agents of a number of diseases in humans. These bacteria are transmitted by arthropods, including ixodid ticks. DNA of several Rickettsia spp. was identified in Ixodes persulcatus ticks, however, the association of Ixodes trianguliceps ticks with Rickettsia spp. is unknown. In our study, blood samples of small mammals (n=108), unfed adult I. persulcatus ticks (n=136), and I. persulcatus (n=12) and I. trianguliceps (n=34) ticks feeding on voles were collected in two I. persulcatus/I. trianguliceps sympatric areas in Western Siberia. Using nested PCR, ticks and blood samples were studied for the presence of Rickettsia spp. Three distinct Rickettsia species were found in ticks, but no Rickettsia species were found in the blood of examined voles. Candidatus Rickettsia tarasevichiae DNA was detected in 89.7% of unfed I. persulcatus, 91.7% of engorged I. persulcatus and 14.7% of I. trianguliceps ticks. Rickettsia helvetica DNA was detected in 5.9% of I. trianguliceps ticks. In addition, a new Rickettsia genetic variant was found in 32.4% of I. trianguliceps ticks. Sequence analysis of the 16S rRNA, gltA, ompA, оmpB and sca4 genes was performed and, in accordance with genetic criteria, a new Rickettsia genetic variant was classified as a new Candidatus Rickettsia species. We propose to name this species Candidatus Rickettsia uralica, according to the territory where this species was initially identified. Candidatus Rickettsia uralica was found to belong to the spotted fever group. The data obtained in this study leads us to propose that Candidatus Rickettsia uralica is associated with I. trianguliceps ticks.

Genetic variability of Anaplasma phagocytophilum in ticks and voles from Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia.

Anaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in different mammals. The presence of A. phagocytophilum was assayed in Ixodes persulcatus, Ixodes trianguliceps ticks and Myodes spp. voles from two I. persulcatus/I. trianguliceps sympatric areas in the Omsk region (Western Siberia, Russia). In total, A. phagocytophilum was found in 42/108 (38.9%) of vole blood samples, 13/34 (38.2%) of I. trianguliceps ticks removed from voles, 1/12 (8.3%) of I. persulcatus removed from voles, and 18/279 (7.2%) of questing I. persulcatus. GroESL operon sequence analysis of positive samples revealed three distinct A. phagocytophilum genetic groups previously identified in ticks and mammals in Russia. Genetic group 1 was found in 6/36 (16.7%) of sequenced positive blood samples; this group was previously revealed in I. persulcatus and Myodes spp. voles in different regions of Russia. Genetic group 2 was found in 30/36 (83.3%) of sequenced positive blood samples and all positive I. trianguliceps; this group was previously revealed only in Myodes spp. voles and common shrews (Sorex araneus) in I. persulcatus/I. trianguliceps sympatric areas in the Northern Ural. Genetic group 3 was found in all positive questing I. persulcatus and one blood sample; this group was previously revealed in I. persulcatus and Siberian chipmunks (Tamias sibiricus). We suppose that I. trianguliceps is the most probable vector for A. phagocytophilum of group 2. Analysis of the msp4 gene, intergenic region DOV1, and some other genetic loci has shown that isolates from different genetic groups significantly differ in all studied loci and that A. phagocytophilum of group 2 is closely related to A. phagocytophilum isolates revealed in voles and I. trianguliceps in Europe. A. phagocytophilum of groups 1 and 2 are the most similar to each other, while A. phagocytophilum of group 3 clusters with European A. phagocytophilum isolates from I. ricinus and various mammalian species.