Large-scale analysis of antigenic diversity of T-cell epitopes in dengue virus

BACKGROUND: Antigenic diversity in dengue virus strains has been studied, but large-scale and detailed systematic analyses have not been reported. In this study, we report a bioinformatics method for analyzing viral antigenic diversity in the context of T-cell mediated immune responses. We applied this method to study the relationship between short-peptide antigenic diversity and protein sequence diversity of dengue virus. We also studied the effects of sequence determinants on viral antigenic diversity. Short peptides, principally 9-mers were studied because they represent the predominant length of binding cores of T-cell epitopes, which are important for formulation of vaccines. RESULTS: Our analysis showed that the number of unique protein sequences required to represent complete antigenic diversity of short peptides in dengue virus is significantly smaller than that required to represent complete protein sequence diversity. Short-peptide antigenic diversity shows an asymptotic relationship to the number of unique protein sequences, indicating that for large sequence sets (~200) the addition of new protein sequences has marginal effect to increasing antigenic diversity. A near-linear relationship was observed between the extent of antigenic diversity and the length of protein sequences, suggesting that, for the practical purpose of vaccine development, antigenic diversity of short peptides from dengue virus can be represented by short regions of sequences (~<100 aa) within viral antigens that are specific targets of immune responses (such as T-cell epitopes specific to particular human leukocyte antigen alleles). CONCLUSION: This study provides evidence that there are limited numbers of antigenic combinations in protein sequence variants of a viral species and that short regions of the viral protein are sufficient to capture antigenic diversity of T-cell epitopes. The approach described herein has direct application to the analysis of other viruses, in particular those that show high diversity and/or rapid evolution, such as influenza A virus and human immunodeficiency virus (HIV)

Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

Background: The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. Methodology/Principal Findings: HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007-2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. Conclusions/Significance: Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus. © 2010 Tan et al

Complete-Proteome Mapping of Human Influenza A Adaptive Mutations: Implications for Human Transmissibility of Zoonotic Strains

BACKGROUND: There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations. CONCLUSIONS: Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear unstable, suggesting low pandemic potential of H5N1 in its current form. In addition, adaptation signatures indicate that pandemic H1N1/09 strain possesses multiple human-transmissibility mutations, though not an unusually high number with respect to swine strains that infected humans in the past. Adaptation signatures provide a novel tool for identifying zoonotic strains with the potential to infect humans

Integrating complex systems science into road safety research and practice, part 1: review of formative concepts

Many of our most persistent public health problems are complex problems. They arise from a web of factors that interact and change over time and may exhibit resistance to intervention efforts. The domain of systems science provides several tools to help injury prevention researchers and practitioners examine deep, complex and persistent problems and identify opportunities to intervene. Using the increase in pedestrian death rates as an example, we provide (1) an accessible overview of how complex systems science approaches can augment established injury prevention frameworks and (2) a straightforward example of how specific systems science tools can deepen understanding, with a goal of ultimately informing action

Integrating complex systems science into road safety research and practice, Part 2: Applying systems tools to the problem of increasing pedestrian death rates

Objectives To provide a specific example of how systems dynamics tools can increase understanding of stakeholder mental models' and generate robust systems-based hypotheses about the escalating problem of rising pedestrian death rates in the USA. Methods We designed and facilitated two group model building (GMB) workshops. Participants generated causal loop diagrams (CLDs) individually and in small groups to explore hypotheses concerning time-dynamic interacting factors underlying the increasing rates of pedestrian deaths. Using a grounded theory approach, research team members synthesised the structures and hypotheses into a single CLD. Results CLDs from the 41 participants indicated four core factors hypothesised to have a direct impact on pedestrian fatalities: pedestrian-vehicle crashes, vehicle speed at the time of the crash, vehicle size/dimensions and emergency response time. Participants diagrammed how actions and reactions impacted these proximal factors over time and led to ripple effects throughout a larger system to generate an increase in pedestrian deaths. Hypothesised contributing mechanisms fell within the following broad categories: community responses; research, policy and industry influence; potential unintended consequences of responses to pedestrian deaths; and the role of sprawl. Conclusions This application of systems science tools suggested several strategies for advancing injury prevention research and practice. The project generated robust hypotheses and advanced stakeholder communication and depth of understanding and engagement in this key issue. The CLD and GMB process detailed in this study provides a concrete example of how systems tools can be adopted and applied to a transportation safety topic

Conservation and Variability of Dengue Virus Proteins: Implications for Vaccine Design

Dengue viruses (DENVs) circulate in nature as a population of 4 distinct types, each with multiple genotypes and variants, and represent an increasing global public health issue with no prophylactic and therapeutic formulations currently available. Viral genomes contain sites that are evolutionarily stable and therefore highly conserved, presumably because changes in these sites have deleterious effects on viral fitness and survival. The identification and characterization of the historical dynamics of these sites in DENV have relevance to several applications such as diagnosis and drug and vaccine development. In this study, we have identified sequence fragments that were conserved across the majority of available DENV sequences, analyzed their historical dynamics, and evaluated their relevance as candidate vaccine targets, using various bioinformatics-based methods and immune assay in human leukocyte antigen (HLA) transgenic mice. This approach provides a framework for large-scale and systematic analysis of other human pathogens

Chronic Nicotine Modifies Skeletal Muscle Na,K-ATPase Activity through Its Interaction with the Nicotinic Acetylcholine Receptor and Phospholemman

Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21–31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (−4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser63 and Ser68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM

Evidence for a narrow structure at W~1.68 GeV in eta photoproduction on the neutron

New results on quasi-free $\eta$ photoproduction on the neutron and proton bound in a deuteron target are presented. The $\gamma n \to \eta n$ quasi-free cross section reveals a bump-like structure which is not seen in the cross section on the proton. This structure may signal the existence of a relatively narrow ($M\sim 1.68$ GeV, $\Gamma \leq 30$ MeV) baryon state.Comment: Replaced with published versio

Mistletoe lectin is not the only cytotoxic component in fermented preparations of Viscum album from white fir (Abies pectinata)

<p>Abstract</p> <p>Background</p> <p>Preparations of mistletoe (<it>Viscum album</it>) are the form of cancer treatment that is most frequently used in the complementary medicine. Previous work has shown that these preparations are able to exert cytotoxic effects on carcinoma cells, the extent of which might be influenced by the host tree species and by the content of mistletoe lectin.</p> <p>Methods</p> <p>Using colorimetric assays, we have now compared the cytotoxic effects of <it>Viscum album </it>preparations (VAPs) obtained from mistletoe growing on oak (<it>Quercus robur </it>and <it>Q. petraea</it>, VAP-Qu), apple tree (<it>Malus domestica</it>,, VAP-M), pine (<it>Pinus sylvestris</it>, VAP-P) or white fir (<it>Abies pectinata</it>, VAP-A), on the <it>in vitro </it>growth of breast and bladder carcinoma cell lines. While MFM-223, KPL-1, MCF-7 and HCC-1937 were the breast carcinoma cell lines chosen, the panel of tested bladder carcinoma cells comprised the T-24, TCC-SUP, UM-UC-3 and J-82 cell lines.</p> <p>Results</p> <p>Each of the VAPs inhibited cell growth, but the extent of this inhibition differed with the preparation and with the cell line. The concentrations of VAP-Qu, VAP-M and VAP-A which led to a 50 % reduction of cell growth (IC₅₀) varied between 0.6 and 0.03 mg/ml. Higher concentrations of VAP-P were required to obtain a comparable effect. Purified mistletoe lectin I (MLI) led to an inhibition of breast carcinoma cell growth at concentrations lower than those of VAPs, but the sensitivity towards purified MLI did not parallel that towards VAPs. Bladder carcinoma cells were in most cases more sensitive to VAPs treatment than breast carcinoma cells. The total mistletoe lectin content was very high in VAP-Qu (54 ng/mg extract), intermediate in VAP-M (25 ng/mg extract), and very low in VAP-P (1.3 ng/mg extract) and in VAP-A (1 ng/mg extract). As to be expected from the low content of mistletoe lectin, VAP-P led to relatively weak cytotoxic effects. Most remarkably, however, the lectin-poor VAP-A revealed a cytotoxic effect comparable to, or even stronger than, that of the lectin-rich VAP-Qu, on all tested bladder and breast carcinoma cell lines.</p> <p>Conclusion</p> <p>The results suggest the existence of cytotoxic components other than mistletoe lectin in VAP-A and reveal an unexpected potential of this preparation for the treatment of breast and bladder cancer.</p