Eleven patients responding successfully to first-line antiretroviral therapy (ART) were investigated for proviral drug resistance mutations (DRMs) in RT by ultra-deep pyrosequencing (UDPS). After molecular typing of the class I alleles A and B, the CTL epitopes in the Gag, Nef and Pol regions of the provirus were sequenced and compared to the reference HXB2 HIV-1 epitopes. They were then matched with the HLA alleles with determination of theoretical affinity (TA). For 3 patients, the results could be compared with an RNA sample of the circulating virus at initiation of therapy. Five out of 11 patients exhibited DRMs by UDPS. The issue is whether a therapeutic switch is relevant in these patients by taking into account the identity of the archived resistance mutations. When the archived CTL epitopes were determined on the basis of the HLA alleles, different patterns were observed. Some epitopes were identical to those reported for the reference with the same TA, while others were mutated with a decrease in TA. In 2 cases, an epitope was observed as a combination of subpopulations at entry and was retrieved as a single population with lower TA at success. With regard to immunological stimulation and given the variability of the archived CTL epitopes, we propose a new concept of curative vaccine based on identification of HIV-1 CTL epitopes after prior sequencing of proviral DNA and matching with HLA class I alleles.
Genotypic resistance testing is recommended to evaluate the susceptibility of HIV to antiretroviral drugs. These tests are based on bulk population sequencing and thus consider only variants representing more than 20% of the viral population, whereas next generation sequencing methods allow detection below this threshold. We aimed to evaluate the potential use of ultradeep pyrosequencing (UDPS) for genotypic resistance testing in clinical routine at the University Hospital of Bordeaux, France. We performed UDPS on reverse transcriptase (RT) from 47 HIV-1 individuals, naive of antiretroviral treatment and for whom genotypic resistance testing was requested for clinical management in 2011-2012. In 8.5% of the patients, only low-frequency variants harboring RT drug resistance mutations were detected raising the question of their clinical significance. Rilpivirine-associated resistance mutations were detected in 19.1% of our population study. To conclude, UDPS could become a routine tool for the evaluation of HIV-infected patients in hospital laboratories.
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