European Journal of Medical Research - Deutsche AIDS ...

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June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 121 und 749/�l and 10 uninfected controls. CD3+CD4+ and CD3+CD8+ cells as wells as PDC were quantified using FACS analysis. Migration, activation, maturation, and endocytosis of PDC were determined by the expression of CCR7, CD80, CD83 and BDCA2 (=CD303) at baseline and 20 hours after PBMC stimulation using different ODNs. In addition, the induction of interferon (IFN)-alpha production was determined by ELISA. Results: The PDC counts in HIV-infected patients were significantly reduced compared to the control group (p=0.02). The expression of the marker for endocytosis (BDCA-2) was significantly downregulated in unstimulated PDC of HIV-infected patients (p=0.001). After stimulation, the other surface markers were upregulated to a similar extent in patients and controls. The three ODN classes were significantly different in their induction of IFN-alpha production (CpG-A > CpG-C > CpG-B), as were the different C-class ODNs (p
122 EUROPEAN JOURNAL OF MEDICAL RESEARCH June 27, 2007 negative persons with an acute CMV infection (N=12). To evaluate CMV-specific immune response lymphocytes were isolated by standard procedures. Interferon-g, Interleukin-2, and Tumor-Necrosis-Factor-a production in lymphocytes following short time stimulation with specific antigens (CMV, PMA-Ionomycin as positive control, and negative control) were measured with intracellular cytokine staining and analysis by FACS. The analysis was done as percentage of CD4+ lymphocytes with specific cytokine-detection vs. those without. In HLA-A2 positive individuals a CMV-specific tetramer staining was performed additionally. Results: Absolute and relative CD4 count was not significant different between the untreated and treated groups. Significant Differences were seen for HIV positive individuals under antiretroviral treatment vs. no treatment for CMV-specific IFN-� production in CD4+ lymphocytes (p < 0,05) but not for Interleukin-2 or Tumor-Necrosis-Factor-a. Also the ratio of Interleukin-2/ Interferon-g were significant different. In the longitudinal study all HIV positive individuals generated after initiation of antiretroviral treatment a significant higher percentage of Interferon-g producing cells than before treatment. In acute CMV infection in HIV negative persons the IL-2/Interferon-g ratio was significant lower compared with the other groups Conclusions: Not only the absolute CD4 cell count is rising during antiretroviral treatment, but also the specific qualitative immune response against recall antigens like CMV is improving as showed here by cytokine production and tetramer staining. F.9 (Poster) Sequence dependent activation of innate immune system by plasmid DNA in a TLR9 dependent and independent manner Kosovac D. 1 , Lütschg V. 1 , Wild J. 1 , Wagner R. 1 1 Universität Regensburg, Institut für Medizinsche Mikrobiologie und Hygiene, Molekulare Mikrobiologie und Gentherapie, Regensburg, Germany Introduction: The main limitation of plasmid-based (pDNA) genetic vaccines is low efficiency in non-human and human primates requiring high amounts of pDNA to properly prime cellular and humoral immune responses. Herein, we tested the influence of vector backbone sequence modifications, mainly CpG-content, on the activation of innate immune system ex vivo in a murine splenocyte model and on human dendritic cells (DCs). Methods and results: Various pcDNA5/FRT (Ref; 100% CpG]) derived vector backgrounds (pDS [50,6% CpG], pDS110- [47,5% CpG]) were synthesised and analysed phenotypically and functionally on murine splenocytes and human plasmacytoid and myeloid DCs. In contrast to Ref and pDS110-, pDS-DNA induces the secretion of high amounts of IFNg and IL-6 after in vitro stimulation of naive mouse splenocytes as demonstrated by cytokine ELISA and ELISPOT analysis. Quantification of secreted cytokines indicated a strong Th1- but not Th2-polarisation by pDS-DNA. Elimination of five CpGs within the 110-region overlapping the prokaryotic origin of replication within pDS or translocation of the 110 bp fragment within the plasmid resulted in a significantly reduced stimulation of proinflammatory cytokines suggesting a strong influence of individual CpGs on induced immune responses in a position dependant manner. Whereas pDS-DNA stimulates splenocytes from wildtype mice to produce high amounts of IFNg and TNFa this effect was totally aborted in TLR9-/- mice. Additionally, ex vivo stimulations of human plasmacytoid dendritic cells with pDS- DNA resulted in secretion of high amounts of type I interferon (IFNa). Confirming the mouse data, deletion of the 110 bp fragment comprising 5 CpG residues (pDS110-) rendered this plasmid immunosilent with respect to the induction of IFNa. Summary: Concerted modification of CpG-amount of the commercial pcDNA5/FRT vector resulted in synthesis of the potent immunogenic DNA expression vector (pDS). Immunogenic properties of pDS, based on CpG content, will make a significant impact for a further rational development of DNA vaccines including HIV vaccine. F.10 (Poster) HIV-1 Vpr induces dysregulation of pDC-mediated IFN-� release by NK cells Hong H. 1 , Bhatnagar N. 1 , Mönkemeyer M. 1 , Schmidt R.E. 1 , Heiken H. 1 1 Medizinische Hochschule Hannover, Klinische Immunologie K14, Hannover, Germany Objective: Natural killer (NK) cells can lyse virus-infected cells or tumour cells without prior sensitization. In addition, they regulate T cell responses by early production of interferon-g (IFN-�). Impairment of NK cell function in HIV-1 infected patients has been extensively studied. However, the exact mechanism of HIV-induced NK cell dysfunction is not yet clear. There has been an increasing interest in the interaction of NK cells with dendritic cells (DCs). Here we addressed the question whether HIV-1 Vpr is able to disturb the interplay between NK cells and plasmacytoid DCs (pDCs). Methods: We sorted NK cells and pDCs from peripheral blood of healthy donors. Highly purified NK cells and pDCs were cocultured and pre-treated with HIV-1 synthetic Vpr before activation with CpG. NK phenotype was assessed by FACS analysis and function by standard 51Cr release assay and IFN-� ELISA. Results: In accordance with previously published data, CpG stimulated pDCs were able to activate NK cells as determined by higher CD69 expression, increased cytolytic activity and robust IFN-� release. HIV-1 Vpr did not impair pDC mediated upregulation of CD69 in NK cells and did not interfere with increased NK killing. However, Vpr substantially decreased pDC induced IFN-� secretion. Conclusion: Vpr-mediated dysregulation of early IFN-� production by NK cells could be of considerable importance in the pathogenesis of HIV infection and warrants further studies. F.11 (Poster) Comparison of effector functions, immune activation, proliferation and apoptosis of HIV-specific CD8 T cells in the chronic phase of infection Vollbrecht T. 1 , Brackmann H. 1 , Henrich N. 1 , Roling J. 1 , Bogner J. 1 , Goebel F.D. 1 , Walker B.D. 2 , Draenert R. 1 1Med. Poliklinik / LMU, München, Germany, 2Partners AIDS Research Center / Massachusetts General Hospital, Charlestown, United States of America Background: CD8 T cell responses are thought to play a pivotal role in the control of acute HIV infection. In the chronic
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