Appearance of Drug Resistance-Associated Mutations in Human Immunodeficiency Virus Type 1 CRF01_AE Integrase Derived from Drug-Naive Thai Patients

C ombination therapy or highly active antiretroviral (ARV) therapy (HAART) with two or more reverse transcriptase (RT) inhibitors and protease (PR) inhibitors (PIs) for human immunodeficiency virus type 1 (HIV-1)-infected patients has achieved durable virological suppression as well as appreciably reducing HIV-1 transmission, morbidity, and mortality associated with HIV-1 disease. However, the emergence of drug-resistant viruses as a result of widespread drug use is currently recognized as one of the major obstacles associated with ARV therapy ¹ ; therefore, a new class of ARV drugs targeting the viral integration process has been developed to deal with multidrug resistance viruses. ² Raltegravir (RAL) (Isentress; Merck, Germany) is a first-strand transfer integrase inhibitor (InSTI) approved in 2007 for clinical use. In addition, elvitegravir (EGV), another InSTI, is currently in phase III clinical trials, while several other integrase inhibitors are also under development. ²

HIV-1 is subdivided into three groups, M (major), O (outlying), and N (new or non-M, non-O), and the major HIV-1 pandemic has been caused by group M viruses. The viruses in group M are further classified into subtypes and circulating recombinant forms (CRFs), which are prevalent in specific geographic regions. While subtype B of HIV-1 is the predominant subtype in the Americas, Europe, and Australia, there is a growing epidemic of non-B subtypes and CRFs in Africa and Asia. ³ ARV drugs have been designed against subtype B viruses, but are believed to retain their activity against other subtypes; however, only limited data are presently available as to how viral diversity among different subtypes and CRFs affects drug susceptibility and resistance. CRF01_AE is one of the major HIV-1 subtypes dominating the global epidemic, and is prevalent throughout Southeast Asia. ³ In particular, CRF01_AE is responsible for more than 95% of infections in Thailand, Cambodia, and Vietnam. ³ A recent study of 1250 integrase genes of various subtypes and CRFs, retrieved from the HIV-1 sequence database (http://www.hiv.lanl.gov/), revealed that higher amino acid diversity was observed in CRF01_AE integrase than in subtype B integrase. ⁴ To study naturally occurring drug resistance-associated mutations in Thai CRF01_AE integrase, we performed a genotypic study on the integrase genes derived from plasma samples from HIV-1-infected, drug-naive patients residing in central Thailand.

Peripheral blood samples were received by the National Institute of Health, Thailand for routine HIV-1 drug resistance genotyping in 2006–2008. One hundred randomly selected plasma samples were subjected to this study with approval from the institutional ethics committee of the Research Institute for Microbial Diseases, Osaka University as well as from the Department of Medical Sciences, Ministry of Public Health of Thailand. Viral RNA was extracted using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany). The HIV-1 integrase gene was then amplified by one-step reverse transcription (RT)-coupled polymerase chain reaction (PCR) using a SuperScript III One-Step RT-PCR System with Platinum Tag DNA polymerase (Invitrogen, Carlsbad, CA), followed by nested PCR using KOD DNA polymerase (Toyobo, Osaka, Japan). The primers, Int-outer-S1, 5′-GTCTACCTGTCATGGGTACC-3′ [corresponding to nucleotide (nt) 4140 to 4159 of reference strain NL4-3 (GenBank accession number AF324493)] and Int-Outer-AS1, 5′-GTGGGATATGTACTTCTGAACTTAC-3′ (nt 5215 to 5191), were used for RT and first PCR, and the primers, Int-Inner-S, 5′-GCACACAAAGGAATTGGAGGAAATGAAC-3′ (nt 4161 to 4188) and Int-Inner-AS, 5′-GGATGCTGGTTTCATAGTGATGTCTA TAAAACC-3′ (nt 5185 to 5153), were used for nested PCR. Sequencing analysis of the PCR-amplified HIV-1 integrase gene was then carried out using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA), and data were assembled using SeqScape v2.5 software (Applied Biosystems). The deduced amino acid sequences were then aligned with the sequence of NL4-3, using the Clustal W algorithm and manual editing.

We successfully examined the nucleotide sequences of 78 PCR-amplified integrase genes by direct sequencing. Phylogenetic tree analysis using the neighbor joining method indicated that 75 integrase genes were classified into CRF01_AE, while three integrase genes were classified into subtype B (data not shown). The nucleotide sequences of the integrase genes have been deposited in the GenBank database under accession numbers HM150808–HM150885. The deduced amino acid sequences of 75 CRF01_AE integrase genes were translated and studied for amino acid substitutions by comparing with the subtype B NL4-3 strain. Many subtype-specific mutations were found in CRF01_AE integrase; namely, more than 80% of samples contained amino acid substitutions, K14R, A21T, G24S, V31I, T112V, T125A, G134N, I135V, D167E, V201I, H216Q, L234I, E278D, and S283G (Table 1). Next, we examined the appearance of primary and secondary mutations associated with drug resistance to integrase inhibitors, RAL, EGV, S-1360, L-708906, L-187810, and other integrase inhibitors (clinical and in vitro data), including H51Y, H55Y, T66A/I/K, L68I/V, V72I, L74A/I/M/S, E92A/Q, Q95K, T97A, T112I, H114Y, S119G/R, F121Y, T125K, A128T, E138A/K, G140A/C/S, Y143R/C/H, Q146K/P, S147G, Q148H/K/R, V151I, S153A/Y, M154I/N, N155H/S, K156N, E157Q, K160D/N, G163K/R, V165I, R166S, E170A, V201I, I203M, T206S, S230N/R, D232N, V249I, R263K, and C280Y. ^{4 –10} The results showed that although no primary mutations were detected, secondary mutations associated with resistance to RAL, EGV and/or other inhibitors, V72I, V165I, V201I, and I203M, were detected in more than 5% of samples (Table 2). In particular, V201I was detected in most samples. In addition, other secondary mutations, T97A, T112I, S153A, M154I, T206S and S230N, were detected in a few samples (Table 2).

Previous genotypic studies on HIV-1 integrase revealed that several secondary mutations associated with resistance to integrase inhibitors, including V72I, L74I, E92Q, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N, were frequently detected in drug-naive patients from many countries including Belgium, Brazil, and United States. ^{11 –14} In addition, such integrase mutations were detected in many subtypes and CRFs of HIV-1 including CRF01_AE viruses. ^{4,13 –15} In these reports, drug resistance-associated mutations were suggested to have appeared due to natural polymorphisms at amino acid positions 72, 74, 97, 112, 119, 125, 128, 138, 151, 153, 154, 155, 156, 157, 163, 165, 201, 203, 206, and 230 of HIV-1 integrase. ^{4,11 –14} According to this information, all secondary mutations detected in this study might be due to natural polymorphism. Nevertheless, we consider that further surveillance studies are necessary to identify drug resistance-associated integrase mutations in HIV-1-infected, drug-naive Thai patients.

Finally, a recent report showed that EGV was somewhat less effective in inhibiting the replication of a CRF01_AE virus compared to viruses of other major HIV-1 subtypes in an in vitro study, ⁶ implying that naturally occurring mutations in CRF01_AE integrase undeniably affect viral drug susceptibility to integrase inhibitors. Therefore, phenotypic studies may be required to elucidate the baseline drug susceptibility of CRF01_AE viruses to integrase inhibitors, prior to their introduction in Southeast Asian countries, including Thailand.