Reads2Type: a web application for rapid microbial taxonomy identification.

BACKGROUND: Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data. RESULTS: Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete genomes. Using a dataset of 1003 whole genome sequenced bacteria from various sequencing platforms, Reads2Type was able to identify the species with 99.5 % accuracy and on the minutes time scale. CONCLUSIONS: In comparison with other tools, Reads2Type offers the advantage of not needing to transfer sequencing files, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html.

Benchmarking of methods for genomic taxonomy.

One of the first issues that emerges when a prokaryotic organism of interest is encountered is the question of what it is--that is, which species it is. The 16S rRNA gene formed the basis of the first method for sequence-based taxonomy and has had a tremendous impact on the field of microbiology. Nevertheless, the method has been found to have a number of shortcomings. In the current study, we trained and benchmarked five methods for whole-genome sequence-based prokaryotic species identification on a common data set of complete genomes: (i) SpeciesFinder, which is based on the complete 16S rRNA gene; (ii) Reads2Type that searches for species-specific 50-mers in either the 16S rRNA gene or the gyrB gene (for the Enterobacteraceae family); (iii) the ribosomal multilocus sequence typing (rMLST) method that samples up to 53 ribosomal genes; (iv) TaxonomyFinder, which is based on species-specific functional protein domain profiles; and finally (v) KmerFinder, which examines the number of cooccurring k-mers (substrings of k nucleotides in DNA sequence data). The performances of the methods were subsequently evaluated on three data sets of short sequence reads or draft genomes from public databases. In total, the evaluation sets constituted sequence data from more than 11,000 isolates covering 159 genera and 243 species. Our results indicate that methods that sample only chromosomal, core genes have difficulties in distinguishing closely related species which only recently diverged. The KmerFinder method had the overall highest accuracy and correctly identified from 93% to 97% of the isolates in the evaluations sets.

Multilocus sequence typing of total-genome-sequenced bacteria.

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides.

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.

Use of a Regression Model to Study Host-Genomic Determinants of Phage Susceptibility in MRSA.

Staphylococcus aureus is a major agent of nosocomial infections. Especially in methicillin-resistant strains, conventional treatment options are limited and expensive, which has fueled a growing interest in phage therapy approaches. We have tested the susceptibility of 207 clinical S. aureus strains to 12 (nine monovalent) different therapeutic phage preparations and subsequently employed linear regression models to estimate the influence of individual host gene families on resistance to phages. Specifically, we used a two-step regression model setup with a preselection step based on gene family enrichment. We show that our models are robust and capture the data's underlying signal by comparing their performance to that of models build on randomized data. In doing so, we have identified 167 gene families that govern phage resistance in our strain set and performed functional analysis on them. This revealed genes of possible prophage or mobile genetic element origin, along with genes involved in restriction-modification and transcription regulators, though the majority were genes of unknown function. This study is a step in the direction of understanding the intricate host-phage relationship in this important pathogen with the outlook to targeted phage therapy applications.

Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction.

BACKGROUND: Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein. It does so by integrating predictions of proteasomal cleavage, TAP transport efficiency, and MHC class I affinity. At least four other methods have been developed recently that likewise attempt to predict CTL epitopes: EpiJen, MAPPP, MHC-pathway, and WAPP. In order to compare the performance of prediction methods, objective benchmarks and standardized performance measures are needed. Here, we develop such large-scale benchmark and corresponding performance measures and report the performance of an updated version 1.2 of NetCTL in comparison with the four other methods. RESULTS: We define a number of performance measures that can handle the different types of output data from the five methods. We use two evaluation datasets consisting of known HIV CTL epitopes and their source proteins. The source proteins are split into all possible 9 mers and except for annotated epitopes; all other 9 mers are considered non-epitopes. In the RANK measure, we compare two methods at a time and count how often each of the methods rank the epitope highest. In another measure, we find the specificity of the methods at three predefined sensitivity values. Lastly, for each method, we calculate the percentage of known epitopes that rank within the 5% peptides with the highest predicted score. CONCLUSION: NetCTL-1.2 is demonstrated to have a higher predictive performance than EpiJen, MAPPP, MHC-pathway, and WAPP on all performance measures. The higher performance of NetCTL-1.2 as compared to EpiJen and MHC-pathway is, however, not statistically significant on all measures. In the large-scale benchmark calculation consisting of 216 known HIV epitopes covering all 12 recognized HLA supertypes, the NetCTL-1.2 method was shown to have a sensitivity among the 5% top-scoring peptides above 0.72. On this dataset, the best of the other methods achieved a sensitivity of 0.64. The NetCTL-1.2 method is available at http://www.cbs.dtu.dk/services/NetCTL. All used datasets are available at http://www.cbs.dtu.dk/suppl/immunology/CTL-1.2.php.

What Can We Learn from a Metagenomic Analysis of a Georgian Bacteriophage Cocktail?

Phage therapy, a practice widespread in Eastern Europe, has untapped potential in the combat against antibiotic-resistant bacterial infections. However, technology transfer to Western medicine is proving challenging. Bioinformatics analysis could help to facilitate this endeavor. In the present study, the Intesti phage cocktail, a key commercial product of the Eliava Institute, Georgia, has been tested on a selection of bacterial strains, sequenced as a metagenomic sample, de novo assembled and analyzed by bioinformatics methods. Furthermore, eight bacterial host strains were infected with the cocktail and the resulting lysates sequenced and compared to the unamplified cocktail. The analysis identified 23 major phage clusters in different abundances in the cocktail, among those clusters related to the ICTV genera T4likevirus, T5likevirus, T7likevirus, Chilikevirus and Twortlikevirus, as well as a cluster that was quite distant to the database sequences and a novel Proteus phage cluster. Examination of the depth of coverage showed the clusters to have different abundances within the cocktail. The cocktail was found to be composed primarily of Myoviridae (35%) and Siphoviridae (32%), with Podoviridae being a minority (15%). No undesirable genes were found.

Targeting of conserved gag-epitopes in early HIV infection is associated with lower plasma viral load and slower CD4(+) T cell depletion.

We aimed to investigate whether the character of the immunodominant HIV-Gag peptide (variable or conserved) targeted by CD8(+) T cells in early HIV infection would influence the quality and quantity of T cell responses, and whether this would affect the rate of disease progression. Treatment-naive HIV-infected study subjects within the OPTIONS cohort at the University of California, San Francisco, were monitored from an estimated 44 days postinfection for up to 6 years. CD8(+) T cells responses targeting HLA-matched HIV-Gag-epitopes were identified and characterized by multicolor flow cytometry. The autologous HIV gag sequences were obtained. We demonstrate that patients targeting a conserved HIV-Gag-epitope in early infection maintained their epitope-specific CD8(+) T cell response throughout the study period. Patients targeting a variable epitope showed decreased immune responses over time, although there was no limitation of the functional profile, and they were likely to target additional variable epitopes. Maintained immune responses to conserved epitopes were associated with no or limited sequence evolution within the targeted epitope. Patients with immune responses targeting conserved epitopes had a significantly lower median viral load over time compared to patients with responses targeting a variable epitope (0.63 log(10) difference). Furthermore, the rate of CD4(+) T cell decline was slower for subjects targeting a conserved epitope (0.85% per month) compared to subjects targeting a variable epitope (1.85% per month). Previous studies have shown that targeting of antigens based on specific HLA types is associated with a better disease course. In this study we show that categorizing epitopes based on their variability is associated with clinical outcome.

The cancer exome generated by alternative mRNA splicing dilutes predicted HLA class I epitope density.

Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I supertype representatives consistently found to contain fewer predicted epitopes compared to normal tissue. We observed a significant difference in amino acid composition between protein sequences associated with normal versus cancer tissue, as transcripts found in cancer are enriched with hydrophilic amino acids. This variation contributes to the observed significant lower likelihood of cancer-specific peptides to be predicted epitopes compared to peptides found in normal tissue.

Impact of injecting drug use on mortality in Danish HIV-infected patients: a nation-wide population-based cohort study.

OBJECTIVES: To estimate the impact of injecting drug use (IDU) on mortality in HIV-infected patients in the highly active antiretroviral therapy (HAART) era. DESIGN: Population-based, nation-wide prospective cohort study in Denmark (the Danish HIV Cohort Study). METHODS: A total of 4578 HIV-infected patients were followed from 1 January 1997 or date of HIV diagnosis. We calculated mortality rates stratified on IDU. One-, 5- and 10-year survival probabilities were estimated by Kaplan-Meier methods, and Cox regression analyses were used to estimate mortality rate ratios (MRR). RESULTS: Of the patients, 484 (10.6%) were categorized as IDUs and 4094 (89.4%) as non-IDUs. IDUs were more likely to be women, Caucasian, hepatitis C virus (HCV) co-infected and younger at baseline; 753 patients died during observation (206 IDUs and 547 non-IDUs). The estimated 10-year survival probabilities were 53.2% [95% confidence interval (CI): 48.1-58.3] in the IDU group and 82.1% (95% CI: 80.7-83.6) in the non-IDU group. IDU as route of HIV infection more than tripled the mortality in HIV-infected patients (MRR: 3.2; 95% CI: 2.7-3.8). Adjusting for potential confounders did not change this estimate substantially. The risk of HIV-related death was not increased in IDUs compared to non-IDUs (MRR 1.1; 95% CI 0.7-1.7). CONCLUSIONS: Although Denmark's health care system is tax paid and antiretroviral therapy is provided free of charge, HIV-infected IDUs still suffer from substantially increased mortality in the HAART era. The increased risk of death seems to be non-HIV-related and is due probably to the well-known risk factors associated with intravenous drug abuse.

HLA class I binding 9mer peptides from influenza A virus induce CD4 T cell responses.

BACKGROUND: Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNgamma ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4(+) or CD8(+) T cells prior to the ELISPOT culture revealed that effectors are either CD4(+) (the majority of reactivities) or CD8(+) T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4(+) T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. CONCLUSIONS/SIGNIFICANCE: HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4(+) T cell responses restricted by HLA-II molecules.

Bacteriology in acute exacerbation of chronic obstructive pulmonary disease in patients admitted to hospital.

We investigated the bacterial flora and antimicrobial sensitivity in sputum from patients admitted to hospital with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in order to recommend the best empirical treatment for these patients. The survey was a retrospective study of all patients admitted to Hvidovre hospital with the diagnosis AECOPD during 2004. A total of 118 patients were included. Microscopy, culture and sensitivity testing investigated their sputums. Clinical and paraclinical features were collected from the patients' files. Among the 118 patients, 59 (50%) had a positive sputum culture. The group with positive cultures had a higher blood neutrophil count (p=0.03). There were no other clinical differences between the groups with and without positive sputum culture. The majority of patients with positive sputum cultures were infected with bacteria other than S. pneumonia, mostly H. influenzae and Moraxella catarrhalis. Patients with low FEV(1)<1.0 litre were more likely to be infected with Pseudomonas aeruginosa (p=0.03). The sensitivity patterns of the bacteria showed that the majority were resistant to penicillin. If antibiotics are initiated empirically for AECOPD we recommend either cefuroxime for intravenous treatment or amoxicillin-clavulanate for oral treatment.

Systematic characterisation of cellular localisation and expression profiles of proteins containing MHC ligands.

BACKGROUND: Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable. METHODOLOGY/PRINCIPAL FINDING: In the present large-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly abundant mRNA were found to be much more likely to be the source of MHC ligands. Of the 2.5% most abundant mRNAs as much as 41% of the proteins encoded by these mRNAs contained MHC class I ligands. For proteins containing MHC class II ligands, the corresponding percentage was 11%. Furthermore, we found that most proteins containing MHC class I ligands were localised to the intracellular parts of the cell including the cytoplasm and nucleus. MHC class II ligand donors were, on the other hand, mostly membrane proteins. CONCLUSIONS/SIGNIFICANCE: The results contribute to the ongoing debate concerning the nature of MHC ligand-containing proteins and can be used to extend the existing methods for MHC ligand predictions by including the source protein's localisation and expression profile. Improving the current methods is important in the growing quest for epitopes that can be used for vaccine or diagnostic purposes, especially when it comes to large DNA viruses and cancer.

Broadly immunogenic HLA class I supertype-restricted elite CTL epitopes recognized in a diverse population infected with different HIV-1 subtypes.

The genetic variations of the HIV-1 virus and its human host constitute major obstacles for obtaining potent HIV-1-specific CTL responses in individuals of diverse ethnic backgrounds infected with different HIV-1 variants. In this study, we developed and used a novel algorithm to select 184 predicted epitopes representing seven different HLA class I supertypes that together constitute a broad coverage of the different HIV-1 strains as well as the human HLA alleles. Of the tested 184 HLA class I-restricted epitopes, 114 were recognized by at least one study subject, and 45 were novel epitopes, not previously described in the HIV-1 immunology database. In addition, we identified 21 "elite" epitopes that induced CTL responses in at least 4 of the 31 patients. A majority (27 of 31) of the study population recognized one or more of these highly immunogenic epitopes. We also found a limited set of 9 epitopes that together induced HIV-1-specific CTL responses in all HIV-1-responsive patients in this study. Our results have important implications for the validation of potent CTL responses and show that the goal for a vaccine candidate in inducing broadly reactive CTL immune responses is attainable.

CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening.

The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation, the latter encompassing 12 different HLA class I supertypes with >99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide-HLA-I interactions were synthesized, tested for peptide-HLA-I interactions in a biochemical assay and for influenza-specific, HLA-I-restricted CTL responses in an IFN-gamma ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-I binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential.