Environmental factors controlling seasonal and spatial variability of zooplankton in thermokarst lakes along a permafrost gradient of Western Siberia.

Humic thermokarst lakes of permafrost peatlands in Western Siberia Lowland (WSL) are major environmental controllers of carbon and nutrient storage in inland waters and greenhouse gases emissions to the atmosphere in the subarctic. In contrast to sizable former research devoted to hydrochemical and hydrobiological (phytoplankton) composition, zooplankton communities of these thermokarst lakes and thaw ponds remain poorly understood, especially along the latitudinal gradient, which is a perfect predictor of permafrost zones. To fill this gap, 69 thermokarst lakes of the WSL were sampled using unprecedented spatial coverage, from continuous to sporadic permafrost zone, in order to assess zooplankton (Cladocera, Copepoda, Rotifera) diversity and abundance across three main open water physiological seasons (spring, summer and autumn). We aimed at assessing the relationship of environmental factors (water column hydrochemistry, nutrients, and phytoplankton parameters) with the abundance and diversity of zooplankton. A total of 74 zooplankton species and taxa were detected, with an average eight taxa per lake/pond. Species richness increased towards the north and reached the maximum in the continuous permafrost zone with 13 species found in this zone only. In contrast, the number of species per waterbody decreased towards the north, which was mainly associated with a decrease in the number of cladocerans. Abundance and diversity of specific zooplankton groups strongly varied across the seasons and permafrost zones. Among the main environmental controllers, Redundancy Analysis revealed that water temperature, lake area, depth, pH, Dissolved Inorganic and Organic Carbon and CO2 concentrations were closely related to zooplankton abundance. Cladocerans were positively related to water temperature during all seasons. Copepods were positively related to depth and lake water pH in all seasons. Rotifers were related to different factors in each season, but were most strongly associated with DOC, depth, CH4, phytoplankton and cladoceran abundance. Under climate warming scenario, considering water temperature increase and permafrost boundary shift northward, one can expect an increase in the diversity and abundance of cladocerans towards the north which can lead to partial disappearance of copepods, especially rare calanoid species.

In silico design of influenza a virus artificial epitope-based T-cell antigens and the evaluation of their immunogenicity in mice.

The polyepitope strategy is promising approach for successfully creating a broadly protective flu vaccine, which targets T-lymphocytes (both CD4+ and CD8+) to recognise the most conserved epitopes of viral proteins. In this study, we employed a computer-aided approach to develop several artificial antigens potentially capable of evoking immune responses to different virus subtypes. These antigens included conservative T-cell epitopes of different influenza A virus proteins. To design epitope-based antigens we used experimentally verified information regarding influenza virus T-cell epitopes from the Immune Epitope Database (IEDB) (http://www.iedb.org). We constructed two "human" and two "murine" variants of polyepitope antigens. Amino acid sequences of target polyepitope antigens were designed using our original TEpredict/PolyCTLDesigner software. Immunogenic and protective features of DNA constructs encoding "murine" target T-cell immunogens were studied in BALB/c mice. We showed that mice groups immunised with a combination of computer-generated "murine" DNA immunogens had a 37.5% survival rate after receiving a lethal dose of either A/California/4/2009 (H1N1) virus or A/Aichi/2/68 (H3N2) virus, while immunisation with live flu H1N1 and H3N2 vaccine strains provided protection against homologous viruses and failed to protect against heterologous viruses. These results demonstrate that mechanisms of cross-protective immunity may be associated with the stimulation of specific T-cell responses. This study demonstrates that our computer-aided approach may be successfully used for rational designing artificial polyepitope antigens capable of inducing virus-specific T-lymphocyte responses and providing partial protection against two different influenza virus subtypes.Communicated by Ramaswamy H. Sarma.

Cationic Polymers for the Delivery of the Ebola DNA Vaccine Encoding Artificial T-Cell Immunogen.

BACKGROUND: According to current data, an effective Ebola virus vaccine should induce both humoral and T-cell immunity. In this work, we focused our efforts on methods for delivering artificial T-cell immunogen in the form of a DNA vaccine, using generation 4 polyamidoamine dendrimers (PAMAM G4) and a polyglucin:spermidine conjugate (PG). METHODS: Optimal conditions were selected for obtaining complexes of previously developed DNA vaccines with cationic polymers. The sizes, mobility and surface charge of the complexes with PG and PAMAM 4G have been determined. The immunogenicity of the obtained vaccine constructs was investigated in BALB/c mice. RESULTS: It was shown that packaging of DNA vaccine constructs both in the PG envelope and the PAMAM 4G envelope results in an increase in their immunogenicity as compared with the group of mice immunized with the of vector plasmid pcDNA3.1 (a negative control). The highest T-cell responses were shown in mice immunized with complexes of DNA vaccines with PG and these responses significantly exceeded those in the groups of animals immunized with both the combination of naked DNAs and the combination DNAs coated with PAMAM 4G. In the group of animals immunized with complexes of the DNA vaccines with PAMAM 4G, no statistical differences were found in the ability to induce T-cell responses, as compared with the group of mice immunized with the combination of naked DNAs. CONCLUSIONS: The PG conjugate can be considered as a promising and safe means to deliver DNA-based vaccines. The use of PAMAM requires further optimization.

Artificial Anti-HIV-1 Immunogen Comprising Epitopes of Broadly Neutralizing Antibodies 2F5, 10E8, and a Peptide Mimic of VRC01 Discontinuous Epitope.

The construction of artificial proteins using conservative B-cell and T-cell epitopes is believed to be a promising approach for a vaccine design against diverse viral infections. This article describes the development of an artificial HIV-1 immunogen using a polyepitope immunogen design strategy. We developed a recombinant protein, referred to as nTBI, that contains epitopes recognized by broadly neutralizing HIV-1 antibodies (bNAbs) combined with Th-epitopes. This is a modified version of a previously designed artificial protein, TBI (T- and B-cell epitopes containing Immunogen), carrying four T- and five B-cell epitopes from HIV-1 Env and Gag proteins. To engineer the nTBI molecule, three B-cell epitopes of the TBI protein were replaced with the epitopes recognized by broadly neutralizing HIV-1 antibodies 10E8, 2F5, and a linear peptide mimic of VRC01 epitope. We showed that immunization of rabbits with the nTBI protein elicited antibodies that recognize HIV-1 proteins and were able to neutralize Env-pseudotyped SF162.LS HIV-1 strain (tier 1). Competition assay revealed that immunization of rabbits with nTBI induced mainly 10E8-like antibodies. Our findings support the use of nTBI protein as an immunogen with predefined favorable antigenic properties.

In silico Designed Ebola Virus T-Cell Multi-Epitope DNA Vaccine Constructions Are Immunogenic in Mice.

Background: The lack of effective vaccines against Ebola virus initiates a search for new approaches to overcoming this problem. The aim of the study was to design artificial polyepitope T-cell immunogens⁻⁻candidate DNA vaccines against Ebola virus and to evaluate their capacity to induce a specific immune response in a laboratory animal model. Method: Design of two artificial polyepitope T-cell immunogens, one of which (EV.CTL) includes cytotoxic and the other (EV.Th)⁻⁻T-helper epitopes of Ebola virus proteins was carried out using original TEpredict/PolyCTLDesigner software. Synthesized genes were cloned in pcDNA3.1 plasmid vector. Target gene expression was estimated by synthesis of specific mRNAs and proteins in cells transfected with recombinant plasmids. Immunogenicity of obtained DNA vaccine constructs was evaluated according to their capacity to induce T-cell response in BALB/c mice using IFNγ ELISpot and ICS. Results: We show that recombinant plasmids pEV.CTL and pEV.Th encoding artificial antigens provide synthesis of corresponding mRNAs and proteins in transfected cells, as well as induce specific responses both to CD4+ and CD8+ T-lymphocytes in immunized animals. Conclusions: The obtained recombinant plasmids can be regarded as promising DNA vaccine candidates in future studies of their capacity to induce cytotoxic and protective responses against Ebola virus.

The search of CAR, AhR, ESRs binding sites in promoters of intronic and intergenic microRNAs.

MicroRNAs (miRNAs) play important roles in the regulation of gene expression at the post-transcriptional level. Many exogenous compounds or xenobiotics may affect microRNA expression. It is a well-established fact that xenobiotics with planar structure like TCDD, benzo(a)pyrene (BP) can bind aryl hydrocarbon receptor (AhR) followed by its nuclear translocation and transcriptional activation of target genes. Another chemically diverse group of xenobiotics including phenobarbital, DDT, can activate the nuclear receptor CAR and in some cases estrogen receptors ESR1 and ESR2. We hypothesized that such chemicals can affect miRNA expression through the activation of AHR, CAR, and ESRs. To prove this statement, we used in silico methods to find DRE, PBEM, ERE potential binding sites for these receptors, respectively. We have predicted AhR, CAR, and ESRs binding sites in 224 rat, 201 mouse, and 232 human promoters of miRNA-coding genes. In addition, we have identified a number of miRNAs with predicted AhR, CAR, and ESRs binding sites that are known as oncogenes and as tumor suppressors. Our results, obtained in silico, open a new strategy for ongoing experimental studies and will contribute to further investigation of epigenetic mechanisms of carcinogenesis.

Novel approaches in polyepitope T-cell vaccine development against HIV-1.

RV144 clinical trial was modestly effective in preventing HIV infection. New alternative approaches are needed to design improved HIV-1 vaccines and their delivery strategies. One of these approaches is construction of synthetic polyepitope HIV-1 immunogen using protective T- and B-cell epitopes that can induce broadly neutralizing antibodies and responses of cytotoxic (CD8(+) CTL) and helpers (CD4(+) Th) T-lymphocytes. This approach seems to be promising for designing of new generation of vaccines against HIV-1, enables in theory to cope with HIV-1 antigenic variability, focuses immune responses on protective determinants and enables to exclude from the vaccine compound that can induce autoantibodies or antibodies enhancing HIV-1 infectivity. Herein, the authors will focus on construction and rational design of polyepitope T-cell HIV-1 immunogens and their delivery, including: advantages and disadvantages of existing T-cell epitope prediction methods; features of organization of polyepitope immunogens, which can generate high-level CD8(+) and CD4(+) T-lymphocyte responses; the strategies to optimize efficient processing, presentation and immunogenicity of polyepitope constructs; original software to design polyepitope immunogens; and delivery vectors as well as mucosal strategies of vaccination. This new knowledge may bring us a one step closer to developing an effective T-cell vaccine against HIV-1, other chronic viral infections and cancer.

PolyCTLDesigner: a computational tool for constructing polyepitope T-cell antigens.

BACKGROUND: Construction of artificial polyepitope antigens is one of the most promising strategies for developing more efficient and safer vaccines evoking T-cell immune responses. Epitope rearrangements and utilization of certain spacer sequences have been proven to greatly influence the immunogenicity of polyepitope constructs. However, despite numerous efforts towards constructing and evaluating artificial polyepitope immunogens as well as despite numerous computational methods elaborated to date for predicting T-cell epitopes, peptides binding to TAP and for antigen processing prediction, only a few computational tools were currently developed for rational design of polyepitope antigens. FINDINGS: Here we present a PolyCTLDesigner program that is intended for constructing polyepitope immunogens. Given a set of either known or predicted T-cell epitopes the program selects N-terminal flanking sequences for each epitope to optimize its binding to TAP (if necessary) and joins resulting oligopeptides into a polyepitope in a way providing efficient liberation of potential epitopes by proteasomal and/or immunoproteasomal processing. And it also tries to minimize the number of non-target junctional epitopes resulting from artificial juxtaposition of target epitopes within the polyepitope. For constructing polyepitopes, PolyCTLDesigner utilizes known amino acid patterns of TAP-binding and proteasomal/immunoproteasomal cleavage specificity together with genetic algorithm and graph theory approaches. The program was implemented using Python programming language and it can be used either interactively or through scripting, which allows users familiar with Python to create custom pipelines. CONCLUSIONS: The developed software realizes a rational approach to designing poly-CTL-epitope antigens and can be used to develop new candidate polyepitope vaccines. The current version of PolyCTLDesigner is integrated with our TEpredict program for predicting T-cell epitopes, and thus it can be used not only for constructing the polyepitope antigens based on preselected sets of T-cell epitopes, but also for predicting cytotoxic and helper T-cell epitopes within selected protein antigens. PolyCTLDesigner is freely available from the project's web site: http://tepredict.sourceforge.net/PolyCTLDesigner.html.

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

BACKGROUND: Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. FINDINGS: De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. CONCLUSIONS: Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Properties of the recombinant TNF-binding proteins from variola, monkeypox, and cowpox viruses are different.

Tumor necrosis factor (TNF), a potent proinflammatory and antiviral cytokine, is a critical extracellular immune regulator targeted by poxviruses through the activity of virus-encoded family of TNF-binding proteins (CrmB, CrmC, CrmD, and CrmE). The only TNF-binding protein from variola virus (VARV), the causative agent of smallpox, infecting exclusively humans, is CrmB. Here we have aligned the amino acid sequences of CrmB proteins from 10 VARV, 14 cowpox virus (CPXV), and 22 monkeypox virus (MPXV) strains. Sequence analyses demonstrated a high homology of these proteins. The regions homologous to cd00185 domain of the TNF receptor family, determining the specificity of ligand-receptor binding, were found in the sequences of CrmB proteins. In addition, a comparative analysis of the C-terminal SECRET domain sequences of CrmB proteins was performed. The differences in the amino acid sequences of these domains characteristic of each particular orthopoxvirus species were detected. It was assumed that the species-specific distinctions between the CrmB proteins might underlie the differences in these physicochemical and biological properties. The individual recombinant proteins VARV-CrmB, MPXV-CrmB, and CPXV-CrmB were synthesized in a baculovirus expression system in insect cells and isolated. Purified VARV-CrmB was detectable as a dimer with a molecular weight of 90 kDa, while MPXV- and CPXV-CrmBs, as monomers when fractioned by non-reducing SDS-PAGE. The CrmB proteins of VARV, MPXV, and CPXV differed in the efficiencies of inhibition of the cytotoxic effects of human, mouse, or rabbit TNFs in L929 mouse fibroblast cell line. Testing of CrmBs in the experimental model of LPS-induced shock using SPF BALB/c mice detected a pronounced protective effect of VARV-CrmB. Thus, our data demonstrated the difference in anti-TNF activities of VARV-, MPXV-, and CPXV-CrmBs and efficiency of VARV-CrmB rather than CPXV- or MPXV-CrmBs against LPS-induced mortality in mice.