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

784 Abstracts 41210-41214

12th World AIDS Conference

41210 Genotypic prevalence of transmitted ZDV- and
ddl-resistant HIV-1 variants and natural polymorphism in
protease coding region of seroconverters
Claudia Balotta, M. Violin, A. Berlusconi, A. Gori, S. Rusconi, M. Galli,
M. Moroni. Sacco Hospital University of Milan, Via Abarassi 7420157, Milan;
2University of Milan Milan, Italy
Background: A survey was performed in Italy ten and six years after ZDV and ddl
usage, respectively, to study the prevalence of drug-associated mutations in primary infection and to investigate the complex interaction between genotypic and
phenotypic changes occurring in both Reverse Transcriptase (RT) and Protease
domains of pol gene in drug-naive HIV-1 infected individuals.
Methods: Thirty-seven HIV-1 infected individuals with documented seroconversion 6 months before inclusion in the study (median: 2 months, min-max: 1-6)
were studied. The enrollment was from January 1994 to March 1997. Genetic
analysis of RT and Protease regions was performed after genomic DNA extraction
from PBMCs and direct sequencing of three nested PCR products by dye-primer
procedure. Phenotypic susceptibility was independently assessed for each drug
(NRTI + PI) from primary isolates.
Results: Four of 36 subjects (11.1%) showed K70R mutation which is associated with a certain degree of resistance. Two individuals (5.5%) had respectively
both K70R and T215Y or K70R and K219Q mutations usually related to severe
phenotypic resistance. Five of these subjects were re-tested at further timepoints: all but one did not show the reversion of ZDV-mutant genotype. In vitro
susceptibility to ZDV was assessed in HIV-1 isolates of four patients showing
ZDV-related mutations at seroconversion. Three of these isolates were sensitive
to ZDV. Of note, one patient, whose RT carried the 70R mutation, expressed a
fully ZDV-resistant isolate. None of the subjects showed ddl-related mutations.
The frequency of spontaneous changes in HIV-1 Protease was analyzed in 33
individuals. Surprisingly, only 3 subjects (9.1%) had no spontaneous mutations
at positions related to Protease Inhibitor (PI) resistance. Eight, 15, 5, and 2
individuals showed respectively 1, 2, 3 and 4 aminoacid substitutions. Noteworthy, several of these mutations are known to be involved in PI drug-resistance
occurrence. Therefore, we tested a panel of antiretrovirals (2 NRTI and 2 PI) in
a patient showing a PI-related multiple mutation pattern. The results indicated a
sensitive phenotype for each of the compounds we tested.
Conclusions: Our data demonstrate that the rate of transmitted ZDV-resistance increases overtime. Mutation-bearing strains show to be still susceptible to
ZDV in in vitro assays. These isolates might respond to future treatment including
ZDV. A large polymorphism of Protease region is present in naive population
and stands for preexisting mutations. Although the only patient we tested for
PI susceptibility resulted sensitive, the biological relevance of natural occurring
mutations needs to be defined by further observations.
41211 In vitro drug susceptibility and sequence analysisof
HIV-1 group M isolates belonging to different subtypes
Diane Descamps, C. Apetrei, G. Collin, I. Lousset-Ajaka, F. Damond, F. Simon,
F. Brun-Vezinet. Laboratoire de Virologie, Hopital Bichat Claude Bernard, 46
Rue Henri Huchard, Paris, France
Objective: To evaluate the genotypic and phenotypic susceptibility of HIV-1 group
M strains to antiretroviral drugs.
Methods: The protease Sand RT regions of 40 HIV-1 strains from naive patients genotyped by HMA as belonging to different env subtypes (12A, 12B,
3C, 2D, 3E, 4F, 3G, 1H) were sequenced and phylogenetically analysed. The
phenotypic susceptibility of cellular isolates was analysed in a PBMC assay,
calculating the IC50/90 values for 7 antiretroviral compounds: zidovudine (ZDV),
TIBO derivative R82913, delavirdine and nevirapine, saquinavir (SQV), ritonavir
(RTV) and indinavir (IDV).
Results: All the strains were susceptible to ZDV. Four strains (1A, 1D, 1F
and 1G) presented reduced susceptibility toward TIBO derivative. The subtype A
resistant strain also displayed natural resistance to the other non-nucleoside RT
inhibitors (NNRTI) tested. A second subtype A strain was resistant to delavirdine.
The RT gene sequences and genotypic analyses are pending. When testing the
susceptibility towards protease inhibitors (PI), all but 4 strains (3 subtype G strains
and one subtype E strain) were susceptible. The analysis of the protease gene
revealed that the SQV-resistant strains (2 subtype G and the subtype E strain)
presented the V821 mutation, already associated with decreased susceptibility to
PI, whereas RTV-resistant subtype G strains (n = 3) harbored mutation C67E,
which has never been described.
Conclusions: Our study revealed that natural reduction in susceptibility to
NNRTI may occurs as a consequence of viral variability. This is particularly important for subtype A isolates resistant to NNRTI, as clinical trials using these
compounds are in progress in African countries where this subtype dominates
the epidemic. Our results reinforce the need for global screening of HIV-1 group
M isolates by sequencing and phenotyping susceptibility before initiation of antiretroviral therapy.

555*/41212 A resistance surmountable HIV protease inhibitor
effective against both wild-type and new analogs
of HIV-1 protease
Yoshiaki Kiso, M. Kishida, H. Matsumoto, S. Mizumoto, T. Kimura. Kyoto
Pharmaceutical University, Yamashinaku, Kyoto 607-8414, Japan
Objectives: To overcome the drug-resistance of HIV, a new HIV protease inhibitor

was designed and examined against synthetic HIV protease analogs.
Methods: The HIV-1 protease analogs were synthesized on solid phase and
the dimer analog covalently linked by disulfide bridge (Fig. 1) was constructed
using chemical ligation method. An HIV protease inhibitor, KNI-241 was designed
based on the substrate-transition state. KNI-241 and some other HIV protease inhibitors were tested using wild-type HIV-1 protease, synthetic protease monomer
and dimer analogs.
H- HIV-1 PR (1-50) -NHCH2CH2-S-CH2CO- [Ala67,95,Cys98] HIV-1 PR (53-99) OH
S-S
H- HIV-1 PR (1-50) -NHCH2CH2-S-CH2CO- [Ala67,95,cys98] HIV-1 PR (53-99).OH

Wild type PR PR analog PR analog
<monomer> <dimer>
Results: The HIV-1 protease analogs effectively cleaved the Tyr-Phe-type
substrate, but had weak affinity to Tyr-Pro-type substrate. Consequently, the
molecular recognition of the analogs differs from the wild-type enzyme. KNI-272,
saquinavir, ritonavir, and indinavir weakly inhibited the enzyme analogs whereas
KNI-241 showed strong inhibition against both wild-type enzyme and synthetic
analogs (Fig.2).
Conclusion: KNI-241 showed strong inhibition against both native enzyme
and synthetic analogs. KNI-241-type compound has the possibility to surmount
drug-resistance of HIV.
41213 1Virologic resistance to efavirenz
Lee Bacheler1, O. Weislow2, S. Snyder2, G. Hanna3. 1Dupont Merck
Pharmaceutical Company, Wilmington, DE; 2SRA Technologies, Rockville, MD;
3Massachusetts General Hospital, Boston, MA, USA
Objective: Efavirenz (Sustiva', DMP 266) is a potent non-nucleoside reverse
transcriptase inhibitor (NNRTI) currently in Phase III clinical development for
reduction of HIV-1 RNA load when used in combination with other antiretroviral
drugs. In order to assess the potential for development of virological resistance
to efavirenz, genotypic and phenotypic characterization of plasma virus and virus
isolates from patients participating in clinical trials of efavirenz was undertaken.
Methods: Nucleic acid sequencing of multiple, independently amplified plasma
virus genomes, isolation and in vitro drug susceptibility testing of viruses from patient PBMCs, and population based sequencing of these replicating virus isolates
were used to characterize resistance to efavirenz.
Results: Nucleic acid sequencing of plasma virus from patients whose viral
load rebounded on efavirenz-containing combination therapy revealed the emergence of K103N RT-mutant viruses around the time of viral load rebound, often
followed by the appearance of viruses with multiple mutations (K 103NN 1081
and K 103N/P225H combinations were the most common). In studies to date
(NNRTI nafve patients), the spectrum of mutations observed in patients who
failed efavirenz-containing combination therapy was not influenced by the other
antiretrovirals used, nor by the dose of efavirenz (200 to 600 mg qd). Virus isolates
from these patients demonstrated phenotypic resistance to efavirenz as well as
resistance to nevirapine and delavirdine. K103N, Y188L and/or V1081 mutations
in the RT gene were found in efavirenz-resistant virus isolates.
Conclusions: K103N was the predominant NNRTI-resistant viral genotype
observed in vivo among efavirenz treatment failures. In most cases, a single nucleotide substitution generated this drug resistant mutant virus, which can emerge
when virus suppression is suboptimal. Post treatment failure virus isolates carrying K103N, V1081 and/or Y188L mutations showed a >20 fold increase in the in
vitro IC50 for efavirenz compared to pre-therapy isolates.
41214 HIV-1 expressing the 3TC-associated M184V mutation in
reverse transcriptase (RT) shows increased sensitivity to
adefovir and PMPA as well as decreased replication
capacity in vitro
Michael Miller, K.E. Anton, A.S. Mulato, P.D. Lamy, N.A. Margot,
J.M. Cherrington. Gilead Sciences, 333 Lakeside Drive Foster City CA 94404,
USA
Background: The 3TC-associated M184V mutation in RT has been shown to
increase the in vitro sensitivity to AZT in AZT-resistant viruses and this can provide
clinical benefit in some patients. In this study, the capacity of the M184V mutation