Institute of Immunology and Genetics

Kaiserslautern, Germany

Institute of Immunology and Genetics

Kaiserslautern, Germany
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Zawada A.M.,Saarland University | Rogacev K.S.,Saarland University | Rotter B.,GenXPro GmbH | Winter P.,GenXPro GmbH | And 3 more authors.
Blood | Year: 2011

Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14 ++CD16 -, intermediate CD14 ++CD16 +, and nonclassical CD14 +CD16 ++ monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14 ++CD16 + monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14 ++CD16 + monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14 ++CD16 + monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14 ++CD16 + monocytes to Ag processing and presentation (eg, CD74, HLA-DR, IFI30, CTSB), to inflammation and monocyte activation (eg, TGFB1, AIF1, PTPN6), and to angiogenesis (eg, TIE2, CD105). In conclusion, we provide genetic evidence for a distinct role of CD14 ++CD16 + monocytes in human immunity. After CD14 ++CD16 + monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity. © 2011 by The American Society of Hematology.


Holcomb C.L.,Hoffmann-La Roche | Hoglund B.,Hoffmann-La Roche | Anderson M.W.,Stanford University | Blake L.A.,Roche Holding AG | And 22 more authors.
Tissue Antigens | Year: 2011

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and conexio atf software allows reliable identification of HLA genotypes at high resolution. © 2011 John Wiley & Sons A/S.


Zagordi O.,University of Zürich | Daumer M.,Institute of Immunology and Genetics | Beisel C.,ETH Zurich | Beerenwinkel N.,ETH Zurich | Beerenwinkel N.,Swiss Institute of Bioinformatics
PLoS ONE | Year: 2012

Recent advancements of sequencing technology have opened up unprecedented opportunities in many application areas. Virus samples can now be sequenced efficiently with very deep coverage to infer the genetic diversity of the underlying virus populations. Several sequencing platforms with different underlying technologies and performance characteristics are available for viral diversity studies. Here, we investigate how the differences between two common platforms provided by 454/Roche and Illumina affect viral diversity estimation and the reconstruction of viral haplotypes. Using a mixture of ten HIV clones sequenced with both platforms and additional simulation experiments, we assessed the trade-off between sequencing coverage, read length, and error rate. For fixed costs, short Illumina reads can be generated at higher coverage and allow for detecting variants at lower frequencies. They can also be sufficient to assess the diversity of the sample if sequences are dissimilar enough, but, in general, assembly of full-length haplotypes is feasible only with the longer 454/Roche reads. The quantitative comparison highlights the advantages and disadvantages of both platforms and provides guidance for the design of viral diversity studies. © 2012 Zagordi et al.


Swenson L.C.,British Columbia Center for Excellence in | Daumer M.,Institute of Immunology and Genetics | Paredes R.,Autonomous University of Barcelona
Current Opinion in HIV and AIDS | Year: 2012

PURPOSE OF REVIEW: Deep sequencing of the V3 region of the HIV envelope gene can detect minority non-R5 variants in patients with high sensitivity and specificity. As next-generation sequencing approaches have matured, the clinical utility of deep sequencing for HIV tropism has entered the clinic. Accurate and sensitive tropism testing is essential for successful treatment with the CCR5 antagonist class of antiretrovirals. RECENT FINDINGS: This review will focus on five aspects of next-generation sequencing for assessing HIV tropism: some background on the necessity of deep sequencing versus other tropism methods; the methodological process of 454 sequencing and analysis; other next-generation sequencing technologies; the diagnostic performance of deep sequencing relative to other tropism assays; and the use of deep sequencing in clinical practice. SUMMARY: This method has emerged quickly as both a research and clinical tool because of its high concordance with commonly used phenotypic tropism assays and its ability to predict virological response to CCR5 antagonist-containing regimens. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.


Daumer M.P.,University of Bonn | Daumer M.P.,Institute of Immunology and Genetics | Schneider B.,University of Bonn | Giesen D.M.,University of Bonn | And 10 more authors.
Medical Microbiology and Immunology | Year: 2011

Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4+ and CD8+ cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable. © 2010 Springer-Verlag.


Verheyen J.,University of Cologne | Schweitzer F.,University of Cologne | Harrer E.G.,Friedrich - Alexander - University, Erlangen - Nuremberg | Knops E.,University of Cologne | And 7 more authors.
Antiviral Therapy | Year: 2010

Introduction: HIV type-1 (HIV-1) protease (PR) and cleavage site (CS) mutations accumulate in protease-inhibitor-resistant isolates. HIV-1 CS mutation 431V is the most frequent treatment-associated CS mutation; however, little is known about its origin in treatment-naive HIV-1 isolates. Recently, it has been shown that the CS mutation 431V is located within the human leukocyte antigen (HLA)-B*13-restricted cytotoxic T- lymphocyte (CTL) epitope RQANFLGKI (RI9). Therefore, we investigated whether the presence of CS mutation 431V might additionally be related to immune escape. Methods: CTL recognition of RI9 and of RI9 variants carrying the 431V or the 436R mutation was analysed by ELISPOT in nine HLA-B*13-positive HIV-1-infected patients. Treatment-naive HIV-1-infected patients with primary drug-resistant HIV-1 isolates (n=58) or carrying 431V (n=4) were genotyped for HLA class I alleles. Results: ELISPOT analysis showed different patterns of CTL recognition of RI9. CS mutation 431V could abrogate recognition by RI9-specific CTL in a subgroup of patients. Nevertheless, in our study, the occurrence of 431V in treatment-naive HIV-1 without primary drug resistance could not be explained by HLA-B*13- mediated immune selection. In patients with primary drug-resistant HIV-1 isolates, the frequency of HLA-B*13 was not increased and HLA-B*13 did not correlate with CS mutations 436R or 431V. Conclusions: HIV-1 CS mutation 431V can abrogate CTL recognition, indicating interactions between development of drug resistance and the CTL response. However, we could not find evidence that the presence of 431V in treatment-naive HIV-1 isolates with and without primary drug resistance is related to immune selection by HLA-B*13 or other HLA class I alleles. ©2010 International Medical Press.


Knops E.,University of Cologne | Daumer M.,Institute of Immunology and Genetics | Awerkiew S.,University of Cologne | Kartashev V.,Rostov State Medical University | And 6 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2010

Objectives: To analyse HIV Gag cleavage site (CS) and non-CS mutations in HIV non-B isolates from patients failing antiretroviral therapy. Patients and methods: Twenty-one HIV isolates were obtained from patients infected with HIV subtype G during an outbreak in Russia 20 years ago. Most patients were failing antiretroviral therapy when genotyping was performed. Results: HIV Gag CS mutations accumulated in protease inhibitor (PI)-resistant HIV isolates and were correlated with the presence of three or more PI resistance mutations. Only 1 of 11 HIV isolates carrying major protease mutations did not harbour treatment-associated CS mutations. Natural polymorphism 453T, often found in HIV non-B subtypes, seems to favour the selection of CS mutation 453I rather than treatment-associated CS mutation 453L. Resistance-associated non-CS mutations (123E and 200I) were also observed in PI-resistant clinical isolates. Non-CS mutations in the frameshift-regulating site, which controls the synthesis of Gag-Pol, did not affect frameshift efficiency in dual luciferase assays. Of note, one of four HIV isolates from patients failing PI therapies without protease mutations harboured Gag mutations associated with PI resistance (123E and 436R) and reverse transcriptase inhibitor mutations conferring resistance to the backbone drug. Conclusions: HIV Gag CS mutations commonly occurred in HIV isolates from patients failing PI therapies and natural polymorphisms at the same position influence their emergence. Non-CS mutations previously associated with PI resistance were also observed in clinical isolates. Gag mutations might indicate the evolution of PI resistance even in the absence of protease mutations. © The Author 2010.


Daumer M.,Institute of Immunology and Genetics | Kaiser R.,University of Cologne | Klein R.,Institute of Immunology and Genetics | Lengauer T.,Max Planck Institute for Informatics | And 3 more authors.
BMC Medical Informatics and Decision Making | Year: 2011

Background: Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage. Methods. Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno [coreceptor]. Results: Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno [coreceptor](10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate. Conclusions: The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities. © 2011 Däumer et al; licensee BioMed Central Ltd.


Thielen A.,Max Planck Institute for Informatics | Thielen A.,Institute of Immunology and Genetics | Lengauer T.,Max Planck Institute for Informatics
Intervirology | Year: 2012

Inferring HIV-1 coreceptor usage from a genotype is becoming more and more important for the appropriate treatment of long-term patients. While results are already encouraging where standard bulk-nucleic acid sequencing methods are used, they are limited with respect to the detection of minor variants. In contrast, next-generation sequencing methods (ultradeep sequencing, pyrosequencing) are capable of sequencing virus quasispecies at very low quantities. However, as well as being very expensive, these methods generate vast amounts of data such that sequence analysis has to be automated by computer assistance. Here, we describe the geno2pheno[454] system which handles all processing and prediction steps involved in the prediction of coreceptor usage from massively parallel sequencing data. The system is split into a JAVA preprocessor which is run locally on the client side and a Web server which generates the prediction results. Predictions are based on the same prediction method as used in the geno2pheno[coreceptor] tool. Copyright © 2012 S. Karger AG, Basel.


PubMed | Institute of Immunology and Genetics
Type: | Journal: BMC medical informatics and decision making | Year: 2011

Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage.Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor].Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate.The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.

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