An operational workflow for producing periodic estimates of species occupancy at national scales.

Policy makers require high-level summaries of biodiversity change. However, deriving such summaries from raw biodiversity data is a complex process involving several intermediary stages. In this paper, we describe an operational workflow for generating annual estimates of species occupancy at national scales from raw species occurrence data, which can be used to construct a range of policy-relevant biodiversity indicators.

Analysis of viral diversity for vaccine target discovery.

Background: Viral vaccine target discovery requires understanding the diversity of both the virus and the human immune system. The readily available and rapidly growing pool of viral sequence data in the public domain enable the identification and characterization of immune targets relevant to adaptive immunity. A systematic bioinformatics approach is necessary to facilitate the analysis of such large datasets for selection of potential candidate vaccine targets.

Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed.

Identification of conserved and HLA promiscuous DENV3 T-cell epitopes.

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4.

Dissecting the dynamics of HIV-1 protein sequence diversity.

The rapid mutation of human immunodeficiency virus-type 1 (HIV-1) and the limited characterization of the composition and incidence of the variant population are major obstacles to the development of an effective HIV-1 vaccine. This issue was addressed by a comprehensive analysis of over 58,000 clade B HIV-1 protein sequences reported over at least 26 years. The sequences were aligned and the 2,874 overlapping nonamer amino acid positions of the viral proteome, each a possible core binding domain for human leukocyte antigen molecules and T-cell receptors, were quantitatively analyzed for four patterns of sequence motifs: (1) "index", the most prevalent sequence; (2) "major" variant, the most common variant sequence; (3) "minor" variants, multiple different sequences, each with an incidence less than that of the major variant; and (4) "unique" variants, each observed only once in the alignment.

West Nile virus T-cell ligand sequences shared with other flaviviruses: a multitude of variant sequences as potential altered peptide ligands.

Phylogenetic relatedness and cocirculation of several major human pathogen flaviviruses are recognized as a possible cause of deleterious immune responses to mixed infection or immunization and call for a greater understanding of the inter-Flavivirus protein homologies. This study focused on the identification of human leukocyte antigen (HLA)-restricted West Nile virus (WNV) T-cell ligands and characterization of their distribution in reported sequence data of WNV and other flaviviruses. H-2-deficient mice transgenic for either A2, A24, B7, DR2, DR3, or DR4 HLA alleles were immunized with overlapping peptides of the WNV proteome, and peptide-specific T-cell activation was measured by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays.

Human leukocyte antigen (HLA) class I restricted epitope discovery in yellow fewer and dengue viruses: importance of HLA binding strength.

Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV epitopes were selected using the EpiSelect algorithm to allow for optimal coverage of viral strains. The selected predicted epitopes were synthesized and approximately 75% were found to bind the predicted restricting HLA molecule with an affinity, K(D), stronger than 500 nM.

Highly conserved influenza A sequences as T cell epitopes-based vaccine targets to address the viral variability.

Vaccines are the only proven effective method for prevention of human infectious diseases. Almost all traditional vaccines require activating immunological memory B cells to secrete neutralizing antibodies against invading pathogens. The complication with influenza viruses is the high viral mutation rate that results in immune escape through modification of the B cell epitopes.

Coexpressed RIG-I agonist enhances humoral immune response to influenza virus DNA vaccine.

Increasing levels of plasmid vector-mediated activation of innate immune signaling pathways is an approach to improve DNA vaccine-induced adaptive immunity for infectious disease and cancer applications. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA (dsRNA) pattern receptor required for innate immune activation in response to viral infection. Activation of RIG-I leads to type I interferon (IFN) and inflammatory cytokine production through interferon promoter stimulator 1 (IPS-1)-mediated activation of interferon regulatory factor 3 (IRF3) and NF-κB signaling.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood.

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects.

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.

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.

Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice.

This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA1*0101, DRB1*0401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1x10(5) immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered.

Comparison of DNA vaccines producing HIV-1 Gag and LAMP/Gag chimera in rhesus macaques reveals antigen-specific T-cell responses with distinct phenotypes.

Optimized DNA expression vectors encoding the native HIV-1 Gag or a fusion of Gag with the lysosomal membrane associated protein 1 (LAMP) were compared for immunogenicity upon intramuscular DNA delivery in rhesus macaques. Both vaccines elicited CD4(+) T-cell responses, but with significant differences in the phenotype of the Gag-specific cells: the native Gag induced CD4(+) responses with a phenotype of central memory-like T cells (CD28(+) CD45RA(-)), whereas the LAMP/Gag chimera induced CD4(+) responses with effector memory phenotype (CD28(-) CD45RA(-)). Antigen-specific T cells producing both IFN-gamma and TNFalpha were found in the animals receiving the native Gag, whereas the LAMP/Gag chimera induced humoral responses faster.

Conservation and variability of West Nile virus proteins.

West Nile virus (WNV) has emerged globally as an increasingly important pathogen for humans and domestic animals. Studies of the evolutionary diversity of the virus over its known history will help to elucidate conserved sites, and characterize their correspondence to other pathogens and their relevance to the immune system. We describe a large-scale analysis of the entire WNV proteome, aimed at identifying and characterizing evolutionarily conserved amino acid sequences.

Conservation and variability of dengue virus proteins: implications for vaccine design.

Background: Genetic variation and rapid evolution are hallmarks of RNA viruses, the result of high mutation rates in RNA replication and selection of mutants that enhance viral adaptation, including the escape from host immune responses. Variability is uneven across the genome because mutations resulting in a deleterious effect on viral fitness are restricted. RNA viruses are thus marked by protein sites permissive to multiple mutations and sites critical to viral structure-function that are evolutionarily robust and highly conserved.

Comprehensive analysis of T cell epitope discovery strategies using 17DD yellow fever virus structural proteins and BALB/c (H2d) mice model.

Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity.

Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation.

We used optimized DNA expression vectors to compare two gene delivery methodologies in rhesus macaques, namely direct DNA injection and in vivo adaptive constant-current electroporation via the intramuscular route. The use of in vivo electroporation increased levels of gene expression and immune responses. We used an optimized HIV gag expression plasmid to show the development of new cellular immune responses in SIV-infected animals controlling viremia.

Hotspot Hunter: a computational system for large-scale screening and selection of candidate immunological hotspots in pathogen proteomes.

Background: T-cell epitopes that promiscuously bind to multiple alleles of a human leukocyte antigen (HLA) supertype are prime targets for development of vaccines and immunotherapies because they are relevant to a large proportion of the human population. The presence of clusters of promiscuous T-cell epitopes, immunological hotspots, has been observed in several antigens. These clusters may be exploited to facilitate the development of epitope-based vaccines by selecting a small number of hotspots that can elicit all of the required T-cell activation functions.

Identification of human-to-human transmissibility factors in PB2 proteins of influenza A by large-scale mutual information analysis.

Background: The identification of mutations that confer unique properties to a pathogen, such as host range, is of fundamental importance in the fight against disease. This paper describes a novel method for identifying amino acid sites that distinguish specific sets of protein sequences, by comparative analysis of matched alignments. The use of mutual information to identify distinctive residues responsible for functional variants makes this approach highly suitable for analyzing large sets of sequences.

Evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets.

Background: Influenza A viruses generate an extreme genetic diversity through point mutation and gene segment exchange, resulting in many new strains that emerge from the animal reservoirs, among which was the recent highly pathogenic H5N1 virus. This genetic diversity also endows these viruses with a dynamic adaptability to their habitats, one result being the rapid selection of genomic variants that resist the immune responses of infected hosts. With the possibility of an influenza A pandemic, a critical need is a vaccine that will recognize and protect against any influenza A pathogen.

In silico characterization of immunogenic epitopes presented by HLA-Cw*0401.

Background: HLA-C locus products are poorly understood in part due to their low expression at the cell surface. Recent data indicate that these molecules serve as major restriction elements for human immunodeficiency virus type 1 (HIV-1) cytotoxic T lymphocyte (CTL) epitopes. We report here a structure-based technique for the prediction of peptides binding to Cw*0401.

A systematic bioinformatics approach for selection of epitope-based vaccine targets.

Epitope-based vaccines provide a new strategy for prophylactic and therapeutic application of pathogen-specific immunity. A critical requirement of this strategy is the identification and selection of T-cell epitopes that act as vaccine targets. This study describes current methodologies for the selection process, with dengue virus as a model system.

Prediction of supertype-specific HLA class I binding peptides using support vector machines.

Experimental approaches for identifying T-cell epitopes are time-consuming, costly and not applicable to the large scale screening. Computer modeling methods can help to minimize the number of experiments required, enable a systematic scanning for candidate major histocompatibility complex (MHC) binding peptides and thus speed up vaccine development. We developed a prediction system based on a novel data representation of peptide/MHC interaction and support vector machines (SVM) for prediction of peptides that promiscuously bind to multiple Human Leukocyte Antigen (HLA, human MHC) alleles belonging to a HLA supertype.