Contribution of Human Immunodeficiency Virus Type 1 Minority Variants to Reduced Drug Susceptibility in Patients on an Integrase Strand Transfer Inhibitor-Based Therapy

Abstract

The role of HIV-1 minority variants on transmission, pathogenesis, and virologic failure to antiretroviral regimens has been explored; however, most studies of low-level HIV-1 drug-resistant variants have focused in single target regions. Here we used a novel HIV-1 genotypic assay based on deep sequencing, DEEPGEN (Gibson et al 2014 Antimicrob Agents Chemother 58∶2167) to simultaneously analyze the presence of minority variants carrying mutations associated with reduced susceptibility to protease (PR), reverse transcriptase (RT), and integrase strand transfer integrase inhibitors (INSTIs), as well as HIV-1 coreceptor tropism. gag-p2/NCp7/p1/p6/pol-PR/RT/INT and env/C2V3 PCR products were obtained from twelve heavily treatment-experienced patients experiencing virologic failure while participating in a 48-week dose-ranging study of elvitegravir (GS-US-183-0105). Deep sequencing results were compared with (i) virological response to treatment, (ii) genotyping based on population sequencing, (iii) phenotyping data using PhenoSense and VIRALARTS, and (iv) HIV-1 coreceptor tropism based on the phenotypic test VERITROP. Most patients failed the antiretroviral regimen with numerous pre-existing mutations in the PR and RT, and additionally newly acquired INSTI-resistance mutations as determined by population sequencing (mean 9.4, 5.3, and 1.4 PI- RTI-, and INSTI-resistance mutations, respectively). Interestingly, since DEEPGEN allows the accurate detection of amino acid substitutions at frequencies as low as 1% of the population, a series of additional drug resistance mutations were detected by deep sequencing (mean 2.5, 1.5, and 0.9, respectively). The presence of these low-abundance HIV-1 variants was associated with drug susceptibility, replicative fitness, and coreceptor tropism determined using sensitive phenotypic assays, enhancing the overall burden of resistance to all four antiretroviral drug classes. Further longitudinal studies based on deep sequencing tests will help to clarify (i) the potential impact of minority HIV-1 drug resistant variants in response to antiretroviral therapy and (ii) the importance of the detection of HIV minority variants in the clinical practice.

Characterization of minority HIV-1 drug resistant variants in the United Kingdom following the verification of a deep sequencing-based HIV-1 genotyping and tropism assay

“…Similar to previous studies [ 19 , 41 , 49 , 61 ], DEEPGEN™ detected all the drug resistance mutations, in all 109 patients, originally identified in each laboratory using Sanger sequencing. More importantly, a total of 280 additional drug resistance mutations were identified in both cohorts of HIV-infected individuals, i.e., mutations below the limit of detection of Sanger sequencing (~ 20%) [ 14 – 18 ] and only detectable using deep sequencing, therefore modifying the Sanger-based HIVdb scores and overall resistance interpretation.…”

“…As described above, DEEPGEN™ is based on deep sequencing viral RNA extracted from plasma samples and optimized to accurately detect minority HIV-1 variants above a 1% frequency level in the HIV-1 population [ 19 ]. Moreover, this methodology is capable of generating over 10,000 HIV-1 sequences (reads) per patient that can be used to analyze inter- and intra-patient HIV-1 genetic diversity [ 11 , 41 , 49 ]. Phylogenetic analyses confirmed the HIV-1 subtype initially determined for each patient-derived virus with the DEEPGEN™ Software Tool Suite v2 and Geno2Pheno.…”

Impaired human immunodeficiency virus type 1 replicative fitness in atypical viremic non-progressor individuals

“…These results were corroborated by calculating intra-patient HIV-1 population diversity, i.e., the highly replication impaired VNP viruses had a more complex virus quasispecies population, most likely as a consequence of trying to evade the host immune response [93], which was highlighted by the significant correlation between replicative fitness and HIV-1 genetic diversity in highly immunogenic regions such as Gag and Gp120 [31, 42, 92]. We and others have described similar results where drug resistant HIV-1 variants with more heterogeneous virus population had lower viral replicative fitness [94, 95]. It is possible that impaired (less replication competent) HIV-1 strains are capable to ascertain a limited host immune response in VNPs, enough to maintain high levels of viremia, resulting in viral evolution but limiting host immune activation that could exacerbate HIV disease, i.e., decrease CD4 + T-cell counts.…”

Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

“…As described above, our HIV-1-genotyping and coreceptor tropism assay (DeepGen) is based on deep sequencing of viral RNA extracted from plasma samples and optimized to accurately detect minority HIV-1 variants above a 1% level in the HIV-1 population (24). In addition to identifying low-level drug-resistant viruses otherwise not detected by Sanger sequencing, this methodology is capable of generating over 10,000 HIV-1 sequences (reads) per patient to analyze intra-and interpatient HIV-1 genetic diversity (58,59). All 65 patient samples were genotyped, generating a total of 7,979,986 quality reads with an average read length of 171 bp.…”