Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

12th World AIDS Conference

Abstracts 41215-41218 785

to alter the in vitro susceptibility to adefovir, a nucleoside RT inhibitor whose
prodrug Preveon" is currently in phase III HIV clinical trials, was investigated.
Methods: During clinical trials investigating the anti-HIV activity of Preveon, a
number of patients developed the M184V mutation due to concomitant 3TC therapy. Recombinant viruses from baseline (M184wt) and post-treatment (M184V)
plasma samples were prepared from 4 patients who had AZT-resistance mutations in RT and from 4 patients with AZT-sensitive viruses. Site-directed recombinant HXB2D HIV-1 expressing the M184V, M1841, T215Y and the double
T215Y/M184V mutations were also constructed. Drug-susceptibility assays and
growth kinetic analyses were performed for these viruses.
Results: Matched patient recombinant pairs from all 4 patients who developed
the M184V mutation in the context of high-level AZT resistance showed 3-4 fold
increases in sensitivity to adefovir resulting in near wild-type IC50 values for
adefovir. Increases in AZT sensitivity were also observed in viruses from all 4
patients, but only those from 2 patients reverted to wild-type AZT values. There
were also 2-3 fold increases in sensitivity to a related nucleotide analog PMPA.
Increases in adefovir and PMPA sensitivity were also detected for the site-directed
HXB2D double mutant T215Y/M184V as compared to the T215Y single mutant.
Among AZT-sensitive clinical isolates, increased adefovir sensitivity was more
variable and ranged from no change to a 3-fold increase. Two-fold increases
in adefovir sensitivity were also noted for the M184V and M1841 site-directed
recombinants, rendering these viruses mildly hypersensitive. In growth kinetics
studies, the M184V mutation resulted in attenuated virus growth in all genetic
backgrounds, including those demonstrating high-level AZT resistance.
Conclusions: These in vitro drug susceptibility assays demonstrate that the
M184V mutation in both AZT-resistant and AZT-sensitive genetic backgrounds
results in increased sensitivity for adefovir to wild-type or near wild-type IC50
values, suggesting that patients with viruses possessing the T215Y and M184V
mutations may derive clinical benefit from Preveon therapy. The reduced in vitro
replication capacity of viruses containing the M184V mutation may also contribute to this benefit. The clinical significance of these in vitro findings is under
investigation.
|41215 1Nelfinavir in HAART-experienced HIV-1 infected patients:
Prediction of response by protease genotyping
Patrizio Lorenzi1, J.P. Chaves2, L. Kaiser3, B. Hirschel3, L. Perrin3, S. Yerly3.
1Division of Infectious Diseases University Hospital, 1211 Geneva 14; 2Chetiin
Porchat 24 1004 Lausanne; 3Swiss HIV Cohort Study; Division of Infectious
Diseases, AIDS Center Geneva, Switzerland
Background: Mutations at codon 30 confer resistance to NELF in vitro. Major
resistance mutations for other PIs are located at codons 46, 48, 82, 84 and 90,
suggesting lack of cross-resistance.
Methods: Pilot study. 33 consecutive compliant HAART-experienced patients
(>3 months) who received NELF plus additional antiretrovirals (physician's choice)
because of failure (HIV RNA - 1,000 copies/ml) of previous HAART. Relation
between virologic outcome (response = drop of viremia >1 log at week 4-8) and
number of PI-resistance (PI-R) mutations at baseline was analysed.
Results are shown in the table.
Baseline values

184 & 215 of the reverse transcriptase gene of HIV-1. We compared the results
between the LiPA HIV-1 RT assay and conventional solid phase DNA sequencing
at codons 41, 69 & 70, 74 & 75, 184, and 215 of the reverse transcriptase gene
of HIV-1 using clinical samples from patients on a variety of clinical regimens and
from a range of geographically disparate locations.
Methods: Five European, one American and one Australian Centre participated in this study. In total 598 clinical samples were amplified and tested by
both LiPA HIV-1 RT and conventional cycle sequencing. For the purposes of this
analysis, the LiPA strip was divided into five distinct codon regions comprising of
codon 41, codon 69 and 70, codon 74 and 75, codon 184 and codon 214 and
215. The results were analysed by comparing LiPA and sequence data for each
independent codon region. The result at each codon region was defined as being
either concordant or discordant with the sequence data.
Results: There was greater than 99.0% concordance observed between the
LiPA and conventional sequencing at the codon regions described.
Conclusions: The LiPA HIV-1 RT assay has shown excellent concordance
with conventional DNA sequencing at the codon regions assessed. We suggest
these observed discordancies may be due to the proven higher sensitivity of
the LiPA test to detect minor populations in a mixture. We conclude that the
assay compares very favourably to sequencing for genotypic analysis of selected
regions within the RT gene, is simple to perform, easy to interpret, and has a
relatively high throughput when compared with conventional DNA sequencing.
S41217 Comparison of protease mutations in HIV-1 genomes
detected in plasma and blood mononuclear cells
Catherine Tamalet1, Nathalie Koch1, N. Yahi1, F. Ariasi2, J. Fantini2. 1Hdpital
Timone Laboratoire Virologie 264, rue St Pierre, Marseille; 2CNRS ESA 6003
Faculte St Jerome, Marseille, France
Objective: To assess whether mutations associated with resistance to protease
inhibitors could be detected in plasma prior to peripheral blood mononuclear cells.
Design: 32 pairs of plasma and mononuclear cells samples were analyzed.
Methods: The samples were extracted for total RNA or DNA and amplified
using a RT/PCR or a PCR amplification protocol. The entire HIV-1 protease gene
was sequenced.
Results: In 47% of cases (15/32 samples), there was no difference in the
protease gene sequenced from plasma and mononuclear cells. In this group, the
number of mutations varied from 1 (8 samples) to 9 (1 sample). The most frequent
mutations were L63P (87%), L101 (27%), A71T (20%), M461 (13%) and L90M
(13%). The remaining 53% samples (17/32) were classified in two subgroups, i.e.
with 1-3 differences (8 samples) and with more than 4 differences (9 samples). In
the first case, mutations associated with resistance to ritonavir or indinavir were
found in only 3 of the 8 samples. In the second subgroup, resistance mutations
to various protease inhibitors were found in all cases. It is worth noting that for
3 samples of this subgroup, the DNA genome displayed a heterozygous pattern
which was no longer observed in RNA.
Conclusion: In this study, 103 mutations (77% of the total number of detected
mutations) were identical in DNA and RNA, among which 51% are associated with
resistance. However, a total of 31 mutations of the protease gene were detected
in RNA but not in DNA, and 71% of these mutations were associated to in vitro
resistance to protease inhibitors. Thus, sequencing HIV-1 RNA genomes from
plasma may allow the early identification of resistance mutations in the protease
gene.
41218 Genotypic changes in HIV RT which develop during
PreveonT (adefovir dipivoxil) therapy do not decrease
susceptibility to PMPA
Michael Miller, J.M. Cherrington, P.D. Lamy, A.S. Mulato, K.E. Anton,
N.A. Margot. Gilead Sciences, 333 Lakeside Drive Foster City CA 94404, USA
Background: Preveon' (adefovir dipivoxil; bis-POM PMEA) and PMPA Prodrug
are both nucleotide analogs currently in clinical trials for the treatment of HIV.
In vitro experiments showed that a K65R or K70E mutation developed in the
presence of adefovir, while the K65R mutation arose under the selective pressure
of PMPA. To determine whether viruses carrying mutations in RT selected by
Preveon in patients would remain susceptible to PMPA, viruses were constructed
expressing RT genes from patients who had received extensive Preveon therapy.
Additionally, samples from SIV-infected monkeys and HIV-infected patients who
had received PMPA therapy were characterized to determine whether resistance
to PMPA arose in either setting.
Methods: Genotypic analyses were conducted on RT-PCR products containing
nucleotides 1-900 of HIV RT generated from patient plasma samples. Phenotypic
analyses were conducted on recombinant viruses constructed by site directed
mutagenesis of HXB2D or generated from patient plasma samples.
Results: Recombinant HIV strains were constructed from all 8 of the 29 patients completing an extended Preveon dosing phase who developed sequence
changes from baseline which were possibly associated with Preveon therapy.
Viruses constructed from 2 patients each who developed either a K70E or T69D

mutation in RT showed no decreased susceptibility to PMPA. The remaining 4
patients had developed AZT-associated resistance mutations in HIV RT; these
viruses showed a <3-fold decrease in PMPA susceptibility compared to matched
pre-therapy viruses. Experiments using site directed HXB2D recombinants expressing the corresponding mutations yielded similar results. We also investigated
the emergence of resistance to PMPA as a result of PMPA treatment in vitro and
in vivo. In vitro the K65R mutation in RT was selected and conferred a 3-fold

CD4 cells (/mm3)
Viremia (log copies/mi)
Major NELF mutation (n*)
Minor NELF mutations') (n)
Other Major mutations2) (n )
Total of PI-R mutations3) (n)

Responders (R) (N = 10) Non-responders (NR) (N
143 (8-429) 115(3-295)
4.53 (3.07-6.04) 5.24 (3.89-6.15)
not detected not detected
1 (0-3) 3 (0-5)
1 (0-2) 2 (0-3)
3 (0-4) 5 (0-8)

23) P4)
0.46
0.09
0.016
0.005
0.003

Values are median and (range). Median number of mutations per patient. 1) Codons 36, 63,
71, 77, 88, 90. 2) Codons 46, 48, 82, 84, 90. 3) Include 1) and 2) plus codons 10, 20, 32, 54.
4) Mann-Whitney test.
No patient developped the D30N mutation, but 5/23 NR and 2/10 R developped
at least one other major PI-R mutation on NELF, whereas 6/23 NR and 3/10 R
developped >1 minor NELF mutation.
Conclusions: Non-Response to rescue therapy with NELF in HAART-experienced patients was frequent overall (23/33 or 70%), but 100% (11/11) in those
with >5 PI-resistance mutations before starting NELF, compared to 69% (9/13)
with 3-5 mutations, and 33% (3/9) in those with 0 to 2 mutations. D30N was
not detected at baseline and during NELF. These data support cross-resistance
between NELF and other currently used PIs.
41216 A comparison of sequencing and a reverse hybridisation
assay (LiPA) for the detection of mutations in the RT gene
of HIV-1
Colin Evans1, D. Parker1, F. Sheridan1, J. Scott1, G. Wouters2, L. Styuver2,
A. Van Den Abeele2. Murex Biotech Limited, Central Road, Dartford, Kent,
England; 2lnnogenetics BV Canadastraat, Zwijndrecht, Antwerp, Belgium
Background: Solid phase DNA sequencing is considered the "gold standard" for
genotypic analysis. Recently the Line probe assay (LiPA) has been developed for
detection of mutations of the reverse transcriptase gene of HIV-1 which have been
associated with resistance to AZT, 3TC, ddl and ddC. The Murex-lnnogenetics
LiPA HIV-1 RT assay offers a simple high throughput system for the detection of
both consensus wild type and mutant genotypes at codons 41, 69, 70, 74, 75,