European Journal of Medical Research - Deutsche AIDS ...

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