European Journal of Medical Research - Deutsche AIDS ...

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