European Journal of Medical Research - Deutsche AIDS ...

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June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 119 Methods: Intra-assay variability for both real time assays (m1000/m2000rt and CAP/CTM) was measured with 10 or 20 replicates of a WHO (NIBSC, UK) and a PEI (Paul-Ehrlich Inst., Germany) standard in different dilutions. Inter-assay variability was measured with dilutions ranging from 50- 5x105 cps/mL of one clinical sample (3x5 repeats). 20 seronegative samples and a subtype panel (A-H; NRZ, Germany) were analyzed with each assay (2 group O isolates only with m1000/m2000rt). 97 clinical plasma samples were tested in duplicates by all assays performed according to manufacturer’s instruction. Hands-on-time was documented. Deming regression and Bland Altman analysis were used for comparison. Results: All HIV-seronegative samples were analysed as negative by each system. All subtypes were detected, group O only by m1000/m2000rt. The coefficients of variation (CV) for the different clinical specimen dilutions were between 2.2% and 13.5% for m1000/m2000rt and 6.9% and 32% for CAP/CTM. Linear dilution magnitude for m1000/m2000rt resulted in values below and for CAP/CTM slightly above the nominal concentration, but both systems ensured linearity and correlation for both standards (m1000/m2000rt: CV absolute 11.7-23.9%; R2 0.93-0.97; CAP/CTM: CV 20-50.3%; R2 0.71-0.89). Correlations (R2) and mean differences (Bland/Altman) for all clinical samples were between 0.94 and 0.98 or 0.10-0.40 Dx log, respectively. A reduced handson-time was calculated for the fully automated CAP/CTM followed by m1000/m2000rt. Conclusions: The assays evaluated were comparable in sensitivity and specificity and can be used to monitor the VL of patients infected with HIV-1 group M isolates. m1000/m2000rt can be used for group O. Nevertheless, the VL values obtained were variable, with a mean difference in VL between 0.1 and 0.4 log depending on the system used. This should be taken into account when monitoring VL of the same person with different systems. F. Immunologie F.1 (Vortrag) Analysis of the sequence of HIV-1-protease and the development of drug resistance in the context of HIV-1 protease-specific T-cells Müller S.M. 1 , Schätz B. 1 , Eismann K. 1 , Bergmann S. 1 , Walter H. 2 , Schmidt B. 2 , Korn K. 2 , Spriewald B. 3 , Schmucker M. 4 , Harrer E.G. 1 , Harrer T. 1 , German Competence Network on HIV/AIDS 1 Dept. of Medicine III, Immunodeficiency Unit, University Hospital, Erlangen, Germany, 2 Inst. of Clin. and Mol. Virology, University of Erlangen, Erlangen, Germany, 3 Dept. of Medicine III, HLA Diagnostics, University Hospital, Erlangen, Germany, 4 Dept. of Psychology, University of Erlangen, Erlangen, Germany Objective: Considering the various pathways of the development of drug resistance against protease inhibitors (PI), it is likely that immunological host factors interact with the emergence of resistance mutations in the HIV-1 protease (PR). To determine the influence of HIV-1-specific cytotoxic T-cells (CTL) on the development of drug resistance mutations in HIV-1 PR, we analyzed PR sequences and PR-specific CTL in a HLA class-I-typed cohort of 94 HIV-1-infected patients. Methods: Correlations between HLA alleles and amino acid substitutions were detected by univariate statistical analyses. T-cell activity was measured by ELISPOT and Cr51-release assays from peptide stimulated T-cell lines. Results: Drug mutations and drug-associated polymorphisms showed associations to HLA class-I alleles. Based on these associations we defined several new CTL epitopes, which we verified by biological assays. The important drug resistance mutations M46I, which is conferring resistance to most commonly used PIs was found to be associated with HLA A2 and HLA B62. M46I is located in an already described A2 epitope. Additionally, we defined a B62 epitope which comprises the mutation. In both epitopes the M46I-mutation could act as CTL escape mutation. However, many A2-positive patients were able to recognize peptides containing the mutation and were able to mounted oligoclonal CTL responses, whereas the M46I acted as escape mutation in most B62-positive patients. Conclusions: Our study demonstrates that the HIV-1-specific CTL response shapes the sequence of the PR. Our analyses suggest that the immunoselection exerted by some HLA alleles are facilitates the development of drug resistance mutations in the HIV-1 PR. This interaction between CTL response and drug resistance mutations may have important clinical implications for to the understanding of drug resistance pathways, the sequencing of PIs in antiretroviral therapy and the design of therapeutic vaccines. F.2 (Vortrag) Impact of impaired processivity of HIV-1 reverse transcriptase on the rate of G to A mutations induced by APOBEC-3F/-3G Knoepfel S.A. 1 , Rauch P. 1 , Salisch N. 1 , Allers K. 1 , Huelsmann P.M. 1 , Metzner K.J. 1 1 Institut für Klinische und Molekulare Virologie, Erlangen, Germany Objective: The cytidine deaminases APOBEC-3F and -3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptidelike 3F/-3G) can inhibit human immunodeficiency virus type-1 (HIV-1) replication by inducing G to A mutations in newly synthesized viral DNA. However, HIV-1 is able to overcome the antiretroviral activity of those enzymes by the viral protein Vif. It is presumable that the amount of G to A mutations in newly synthesized viral DNA depends on the time in which the HIV-1 DNA remains single-stranded during reverse transcription. We hypothesize that RTs differing in their processivities and subsequently in their rates of DNA synthesis will have an impact on the rate of G to A mutations introduced by APOBEC-3F/-3G. To prove this, we performed experiments with HIV-1 wild-type (wt) and the M184V- and M184I-variants which confer high resistance against lamivudine and are increasingly impaired in their processivities, respectively, in peripheral blood mononuclear cells (PBMC) but not in T cell lines. Methods: The different RT-variants were cloned both into pNL4-3(-)vif and pNL4-3(+)vif. 293T-cells were transfected, 48h later supernatants were DNase treated and used to infect the H9 T-cell line and PBMCs. After 36h, supernatants were transferred to uninfected cells. Additional 6h later, those cells were harvested, DNA was isolated, and part of env-gene was amplified, cloned, and sequenced. The Mann-Whitney test was used for statistical analysis. Results: Sequences derived form env-clones of HIV-1 NL4- 3(+)vif-wt/M184V/M184I exhibited no G to A hypermutation
120 EUROPEAN JOURNAL OF MEDICAL RESEARCH June 27, 2007 independent of the cells used. In H9 T-cells, increased levels of G to A mutations were detected using HIV-1 NL4-3(-)vif, but no significant differences in the amount of G to A mutations was observed in regard to the different RTs. However, increased amount of G to A mutations were seen in viral DNA of PBMCs infected with HIV-1 NL4-3(-)vif-M184I compared with wt- or M184V-RT variants. Conclusion: High reduction of the rate of DNA synthesis (M184I-RT) leads to an increase of the amount of G to A mutations induced by APOBEC-3F/-3G whereas moderate reduction of processivity (M184V) has no effect on the amount of G to A mutations suggesting that APOBEC-3F/-G induces G to A mutations in a time-dependent manner. F.3 (Poster) Peptide HIV p24 aa14-22-mediated HLA-E stabilization alters cross-talk between natural killer cells and dendritic cells Schulte D. 1 , Körner C. 1 , Krämer B. 1 , Vogel M. 1 , Langhans B. 1 , Nischalke H.D. 1 , Sauerbruch T. 1 , Spengler U. 1 , Rockstroh J.K. 1 , Nattermann J. 1 1 Uni-Klinikum Bonn, Medizinische Klinik I, Bonn, Germany Introduction: Recently, we demonstrated the HIV peptide p24 aa14-22 (PEP-I) to bind to HLA-E, the natural ligand of the inhibiting natural killer (NK) cell receptos NKG2A. Of note, PEP-I-mediated up-regulation of HLA-E resulted in impaired cytotoxic activity of natural killer cells. Here, we analysed whether this interaction alters cytokine secretion of NK cells and affect the maturation and differentiation of dendritic cells. Methods: NK cells from HIV-infected patients and healthy donors were co-cultured with HLA-E-transfected K-562 cells (HLA-E-K562 cells) loaded with and without 10M PEP-I. Monocyte-derived dendritic cells (MO-DC) were incubated with supernatants from these co-cultures and flowcytometrically analyzed for expression of the maturation markers CD83, MHC class I, CD40 and HLA-DR. Results: Both in HIV-infected patients and healthy controls the interaction of PEP-I with HLA-E selectively reduced secretion of TNFa and IFNg but enhanced secretion of IL-6 in those NK cells which had been exposed to HLA-E-K562 cells together with PEP-I. Exposure of MO-DC to supernatants from NK cells stimulated by HLA-E-K562 cells plus PEP-I resulted in a reduced expression of the maturation markers CD40, MHC class I and HLA-DR as compared to MO-DC exposed to NK cell supernatants cultured without PEP-I. Conclusion: Here, we show peptide HIV p24 aa14-22-induced HLA-E stabilization alters cytokine secretion of natural killer cells thereby affecting the cross-talk between NK cells and dendritic cells via soluble mediators. Thus, the interaction between peptide and HLA-E affects further immunoregulatory functions beyond its direct inhibition of NK cell cytolytic activity. F.4 (Vortrag) Characterisation of effector memory phenotypes and CD57-expression on HIV-specific CD8+ T cells Meyer-Olson D. 1 , Simons B.C. 2 , Conrad J.A. 2 , Barnett L. 3 , Lorey S. 3 , Smith R.M. 3 , Schmidt R.E. 1 , Kalams S.A. 3 , Kompetenznetz HIV/BMBF 1 Medizinische Hochschule Hannover, Klinische Immunologie, Hannover, Germany, 2 Vanderbilt University Medical Center, Department of Microbiology and Immunology, Nashville, United States of America, 3 Vanderbilt University Medical Center, Infectious Diseases Unit, Nashville, United States of America Alterations of effector memory phenotype markers such as CD45RA+/CCR7- and markers associated with terminal CD8+ T cell differentiation such as CD57 are a hallmark of chronic HIV-infection, but the structural and functional basis of these changes remain elusive. Differentiation into effector memory phenotype CD8+ T cells with expression of CD45RA (TemRA) is known to be impaired on HIV-specific CD8+ T cells and CD45RO-positive (TemRO) HIV-specific CD8+ T cells are overrepresented in the majority of HIVseropositive subjects. Here, we demonstrate that expansions of terminally differentiated HIV-specific TemRA-phenotype cells can be infrequently detected in HIV-controllers as well as in subjects with higher viral loads. Differentiation into TemRA-phenotype on HIV-specific CD8+ T cells was frequently restricted to distinct clonotypes within an epitopespecific T cell receptor (TCR) repertoire. Expression of CD57 on TemRA HIV-specific CD8+ T cells was not significantly increased as compared to HIV-specific TemRO-phenotype cells but we observed a significant reduction of CD57 expression on HIV-specific TemRA phenotype cells as compared to unselected CD8+ T cells with a TemRA phenotype. Our data provide evidence for structural constraints in HIV-specific CD8+ T cell effector-memory maturation in chronic HIV-infection with distinct expressions of markers associated with terminal differentiation. F.5 (Vortrag) Optimized class C oligodeoxynucleotides (ODN) support the impaired innate immune response of plasmacytoid dendritic cells (PDC) in HIV-infected patients Donhauser N. 1 , Helm M. 2 , Schuster P. 1 , Haupt S. 1 , Vollmer J. 3 , Schmidt B. 1 1 Virological Institute, Clinical and Molecular Virology, Universitätsklinikum Erlangen, German National Reference Centre for Retroviruses, Erlangen, Germany, 2 Praxis Abelein / Helm, Nürnberg, Germany, 3 Coley Pharmaceutical GmbH, Langenfeld, Germany Objective: Human plasmacytoid dendritic cells (PDC) are the major type I-interferon producing cells upon stimulation with CpG oligodeoxynucleotides (ODNs). Patients with human immunodeficiency virus (HIV) infection show a decrease in PDC numbers and a functional impairment. CpG ODNs appear to be attractive therapeutics in HIV, because they may enhance the impaired innate immune response, which may subsequently contribute to the control of virus replication. Methods: The following study was performed using peripheral blood mononuclear cells (PBMC) obtained from 15 untreated HIV-infected patients with CD4+ cell counts between 152
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