17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

CLONAL ANALYSIS OF PROTEASE AND GAG SEQUENCES FROM HIV-1 SUBTYPE C INFECTED PATIENTS FAILING A PROTEASE INHIBITOR

Antivir Ther. 2008; 13(Suppl. 3):A50 (abstract no. 45)

J Ledwaba, V Pillay and L Morris
AIDS Virus Research Unit, National Institute for Communicable Diseases, Sandringham, South Africa

BACKGROUND: High levels of resistance to protease inhibitors (PIs) have been observed both in vitro and in vivo as a result of mutations in the protease (PR) gene. However, compensatory mutations in the p7/p1 or p1/p6 gag cleavage sites have been shown to restore or improve processing efficiency of the resistant enzyme. In this study we examined minority populations in PR and the two gag cleavage sites in patients who had failed Kaletra therapy.

METHODS: The HIV-1 PR gene and the last 174 amino acids of the gag gene were PCR-amplified from four patients failing Kaletra. Population sequencing demonstrated PR resistance in two of these patients (DR108 and DR112), but wild-type sequences in two other patients (DR106 and DR107). The products were cloned into TOPO TA cloning kit. Clonal sequences were aligned and manually edited using Sequencher 4.5. Phylogenetic analysis of the nucleic acid sequences was performed using Mega 3.1.

RESULTS: Analysis of 100 clonal sequences from DR106 showed that 80% were wild-type in both the PR and gag cleavage sites. Polymorphisms in PR were detected in 17% of clones some of which were thought to be intermediate changes. Three individual clones (DR106.07, DR106.51 and DR106.107) had V82A, N88D and D30N PR mutations, respectively. V82A and D30N are well known PR mutations while N88D facilitates the co-occurrence of D30N and L90M. The PR from DR107 was wild-type by population sequencing and one clone (1%) had the V82A mutation by clonal analysis. This isolate contained the K436R polymorphism at the gag cleavage site by population sequencing and on clonal analysis was also shown to have the I437V mutation in the p7/p1 gag cleavage site in 2% of clones. DR108 showed PI resistance mutations M46I, I54V, V82A, I84V and A431V at the p7/p1 gag cleavage site by population sequencing. All clones showed an identical genotype, although one clone (1%) had an additional mutation (I437T) at the p7/p1 site. No other additional major mutations were found in the PR region by clonal sequencing. DR112 showed mutation M46I and no mutations at the gag cleavage sites by population sequencing. All clones were also wild type at both p7/p1 and p1/p6 cleavage sites. Ninety-five percent of clones (95/100) had the mutation M46I and five (5%) were wild type. One clone (1%) (DR 112.09) had additional mutation I84V in the PR region.

CONCLUSION: Population sequencing underestimates the diversity of PI resistance mutations within minority populations following Kaletra administration. Mutations found in the gag cleavage sites are likely to compensate the mutated PR, but might also contribute to PI resistance in those patients who have wild-type PR.

2008-06-10
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