Clonal resistance analyses of HIV type-1 after failure of therapy with didanosine, lamivudine and tenofovir.

BACKGROUND: The rapid failure of initial therapy with combinations of nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) that exclude zidovudine has not been fully explained by standard virus population analyses of HIV type-1 (HIV-1) drug resistance. We therefore investigated HIV-1 genotype and phenotype at the single genome level in samples from patients on a failing regimen of tenofovir (TNV), didanosine (ddI) and lamivudine (3TC). METHODS: Single genome sequencing was performed on 9 failure samples containing both K65R and M184V mutations by standard genotype, either as wild-type/mutant mixtures (6/9) or as mutant only (3/9). Recombinant clones with different combinations of observed mutations were generated and tested for NRTI susceptibility. RESULTS: Of the 204 single genome sequences analysed, 50% were K65R/M184V double mutants, 38% were M184V single mutants, 10% were M184I single mutants and only 1% (2 sequences) were K65R single mutants. Phenotypic testing of recombinant clones showed a significant increase in resistance for double mutants: mean fold resistance to abacavir, ddI and TNV was 6.5, 4.3 and 1.6 for K65R/M184V double mutants versus 2.5, 1.9 and 0.6 for M184V single mutants, respectively (P<0.001). CONCLUSIONS: Mutants with K65R and M184V linked on the same genome were the most common HIV-1 variants in samples analysed from patients failing TNV, ddI and 3TC with both mutations detected by standard genotype. The double mutant exhibited reduced susceptibility to all three NRTIs in the regimen. This resistant phenotype, resulting from just two linked point mutations, likely contributes to rapid failure of NRTI combinations that exclude zidovudine.

Detection of nonnucleoside reverse-transcriptase inhibitor-resistant HIV-1 after discontinuation of virologically suppressive antiretroviral therapy.

Using standard and ultrasensitive techniques, we detected nonnucleoside reverse-transcriptase inhibitor-associated resistance mutations in 11 (20%) of 54 subjects who discontinued virologically suppressive nonnucleoside reverse-transcriptase inhibitor-containing antiretroviral therapy. Resistance was detected in 45% and 14% of subjects with a baseline human immunodeficiency virus type 1 RNA level of 51-400 copies/mL and

Antagonism between the HIV-1 reverse-transcriptase mutation K65R and thymidine-analogue mutations at the genomic level.

Prior virologic and biochemical studies have shown phenotypic antagonism between K65R and multiple thymidine-analogue mutations (TAMs) in site-directed mutants tested in vitro. We hypothesized, on the basis of this observed antagonism, that K65R and T215Y/F with multiple TAMs would not be selected on the same human immunodeficiency virus type 1 genome in vivo. We searched a large database of patient genotypes (n=59,262) for the frequency of K65R in combination with >or=3 TAMs as determined by standard population sequencing. K65R and multiple TAMs were rarely detected (<0.1%) in the same plasma sample. Samples with both K65R and >or=3 TAMs (n=21) were further analyzed by use of single-genome sequencing. K65R was never found on the same genome with T215F/Y and >or=2 other TAMs, except in the presence of the Q151M multiple nucleoside reverse-transcriptase inhibitor (NRTI)--resistance complex. These results indicate that antagonism between the K65R and T215Y/F pathways of NRTI resistance occurs at the genomic level. Therapy with NRTI combinations that select both pathways simultaneously may delay the emergence of NRTI resistance and prolong treatment response.