HIV-minority variants associated with drug resstance to reverse transcriptase and integrase inhibitors and genetic barrier for the development of resistance to integrase inhibitors

Abstract

Minority HIV-1 drug resistance has not been studied in Thailand. Two group of patients, whose conventional genotyping results showed no drug resistance-associated mutations, were investigated: 104 homosexual men recently infected with HIV-1, naїve to antiretroviral treatment and 22 first-line NNRTI-based failures. Pyrosequencing assay was developed to detect and quantify minority Y181C and M184V variants from the patients’ plasma samples. 1/104 (0.96%) and 3/101 (3%) samples were found harboring Y181C and M184V in the group of homosexual men. In patients with first-line treatment failure, one harbored minority M184V mutants (4.5%). Thus, due to such a low prevalence, minority drug resistance test may not be cost-effective for implementing in Thailand. The mechanism of raltegravir (RAL)-resistant evolutions has not been completely elucidated. Because of the emergence of RAL resistance usually initiated with the N155H mutant, we assessed the role of minority N155H-mutated variants in circulating RNA and archived DNA in 5 heavily treated patients experiencing RAL failure and harboring 3 different resistance profiles. No minority N155H-mutated variant was found by allele specific PCR (AS-PCR) in both plasma and whole blood samples collected at baseline and after RAL withdrawal in all 5 patients. During RAL failure, the mutation N155H was detected at different levels in 3 patients displaying the N155H pathway and gradually declined when the double mutant Q148H+G140S was selected in one patient. In two patients with the Q148H resistance pathway, no N155H variant was identified by AS-PCR in both viral RNA and DNA. The N155H mutants do not play a role in determining different resistance profiles. The genetic barrier for the evolution of integrase inhibitors (INIs) including RAL, elvitegravir (EVG), and dolutegravir (DTG) resistance was compared between HIV-1 subtypes B and CRF01_AE by analyzing of 66 substitutions associated with INI resistance at 41 amino acid positions in 144 nucleotide sequences (109 HIV-1 subtype CRFO1_AE and 35 HIV-1 subtype B) of IN gene derived from INI-naїve patients. Most studied amino acid positions including all corresponding to RAL and EVG primary mutations show a high degree of conservation, indication the same genetic barrier between subtypes CRF01_AE and B. Nevertheless, different genetic barriers were observed in two mutations described to be associated with DTG resistance (L101I, A124T) and other five RAL and EVG secondary mutations (V72I, T125K, G140C/S, V201I), which could have an impact on the development of resistance to RAL, EVG, and DTG.