Institut für medizinische Mikrobiologie Basel

1. gp 120 gp 41 -G-I-V- -D-I-V- -G-I-A- -D-I-A- I. Infection experiment with Env-gp41 mutants Literature describes several point mutations in the HIV-1 gene gp41 in patients treated with T-20 that are associated with decreased clinical activity. Accordingly, the motif –D35I -V- (found as GIV in reference virus HXB2) was point mutated to GIV, GIA or DIA in pNL4-3. Mutated Env sequences were used to reconstitute infectious HIV-1 clones, and expression was first quantitatively assessed (quantitative PCR) after in-vitro infection ofthe human lymphocytic cell line CEM-SS. The results showing replication of the different engineered mutants depicted in Figure 1 support the concept that discrete mutations do have an impact on replication capacity (i.e. Fitness). The low replication capacities of mutants however did not allow to assess subtle differences in T-20 susceptibility experiments (not shown). Location of mutants used in the study Figure 1. Replication of Env gp41 mutant in infection assay. II. Comparison of features of replicative vs. non-replicative assay format. We next wanted to check whether the replicative system PhenoTectTM (depicted in Figure 2) that is routinely used for phenotypic diagnosis of HIV resistance of Protease and RT-genes was directly adaptable to assess variations on Env gene by transfection of the four constructs described above. PhenoTect was performed in comparison with a cellular system in which fusion is directly scored without amplification step (non-replicative; Figure 2). The reporter system used in both assays allows to score viral replication as induced b-gal activity which is reported in figure 3 (results of triplicate experiments). Figure 4 depicts the relative percentage of read-out amongst the different variant at day 3 of cultivation and compared to the infection experiment (grey bars). The histogram shows that both replicative and non-replicative methods produce comparable results where dynamics of the virus is less affected than in infection but still reflects the lower fitness of Env-gp41 mutants. III. Susceptibility to T-20 amongst engineered Env-gp41 mutants. We then evaluated the T-20- susceptibilty of the Env-gp41-mutants: GIV, DIV, GIA and DIA in the two reporter systems. Triplicate experiments were performed using either the non-replicative (Figure 5) or the replicative (PhenoTect, Figure 6) format. Results from reporter read-outs were averaged, normalized and curve- fitted. Percentage of virus inhibiton is expressed as a function of T-20 concentration. IC50 values were extrapolated for the two methods as shown if Figures 5 and 6. Figure 7. Figure 6. Figure 5. IV. Micro-study on T-20 susceptibility amongst clinical Env-variants. The Env-gene from the viruses of 8 random patients, all T-20-naïve, was analyzed with the replicative PhenoTect system. IC50 determinations for each one are compiled in the graph shown in Figure 8. The susceptibility plot of Figure 9 compares the relative drug-sensitivity in relation to clonal reference viruses (identical genomes except for the indicated change in gp41). Bars below the line indicate a tendency towards hyper-susceptibility, whereas bars above indicate a tendency towards T-20-resistance. The results emphasize existing heterogeneities in susceptibility among viruses that cannot be predicted from genomic analysis of the “GIV-motifs”. These findings rather indicate that the basis for resistance to the new drug T-20 certainly involves a more complex genetic picture, which is directly deciphered by replicative phenotypic analysis, such as PhenoTect. Reference clone Reference Clone DIV Both non-replicative and Replicative System (PhenoTect) are able to determine fitness features. A replicative System is superior in discerning susceptibilities to fusion inhibitors hence is amenable to be used in diagnosing resistance/susceptibility to fusion inhibitors. Individual mutations may insufficiently predict phenotypic susceptibility to fusion inhibitors. Even drug-naïve patients may need phenotypic analysis for susceptibility to fusion inhibitors. CONCLUSIONS: Figure 8. Figure 9. Quantitative Assessment of Resistance to Fusion Inhibitors (T-20) in a Replicative Phenotyping Assay POSTER 2.22 Institut für medizinische Mikrobiologie Basel Vincent BRONDANI, François HAMY# and Thomas KLIMKAIT Institute of Medical Microbiology, University of Basel, Switzerland # InPheno AG, Basel, Switzerland BACKGROUND: Fusion between HIV-env and target cells is now accepted as a valid target for therapeutic intervention. Nevertheless, like other drugs used in HAART, this new class of fusion inhibitors can experience a rapid escape of HIV via rather stochastic mutations of the HIV genome with subsequent selection upon drug-pressure. We have developed the replicative phenotyping system “PhenoTect”, validated as diagnostic resistance test platform for protease and RT inhibitors. We aimed at assessing the amenability of PhenoTect to analyse resistance against fusion inhibitors. Figure 7 shows a direct comparison of IC50 values determined either in the single cycle (blue bars) or the PhenoTect format (red bars). Results demonstrate the superiority of the replicative format in discriminating susceptibility to T-20 for the examined gp41-mutants.

2. gp 120 gp 41 -G-I-V- -D-I-V- -G-I-A- -D-I-A- Env Or Mutated-Env NON-REPLICATIVE Figure 4. Comparison of Virus dynamics of Env mutants in 3 systems REPLICATIVE Proviral cassette (pNL4-3 background) stable HeLa expressing CD4/CXCR4/CCR5 and LTR-lacZ (Reporter) stable HeLa expressing CD4/CXCR4/CCR5 and LTR-lacZ (Reporter) HeLa (production) Figure 2. Schematical representation of PhenoTectTM (replicative) cellular assay and its non-replicative equivalent. b-Gal b-Gal Figure 3. ONPG HeLa (production) ONPG CEM-SS Lymphocytes (amplification) ONP ONP