Dynamics of Rilpivirine Resistance-Associated Mutation: E138 in Reverse Transcriptase among Antiretroviral-Naive HIV-1-Infected Individuals in Turkey

Introduction

HIV remains a significant global public health problem. According to the World Health Organization (WHO), 37.7 million people will have reported living with HIV by 2020. Among these people, 73% successfully received antiretroviral therapy (ART) to manage their disease. ¹ For Turkey, the situation is similar in that HIV continues to be a health issue for the Turkish public. Turkey's Ministry of Health has declared that from 1985 to November 2021, 29,284 people were diagnosed with HIV. ² According to the International Pharmaceutical Market Database's October 2021 cumulative data, 21,300 HIV-positive individuals received ART in Turkey. ³

The first treatment for HIV was introduced in 1987 worldwide with the discovery of zidovudine (Azidothymidine), and the HIV treatment was developed to ART in 1997, which has become the HIV treatment standard since then. ⁴ Current guidelines for ART in treating HIV consist of at least three antiretroviral drugs to maximize the suppression of HIV and slow the progression to the most advanced stage of the disease—AIDS. The three antiretroviral drugs should be from at least two classes, which are nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase strand transfer inhibitors (INSTIs), fusion inhibitors, protease inhibitors, attachment inhibitors, CCR5 antagonists, postattachment inhibitors, and pharmacokinetic enhancers. ⁵

Recently, this paradigm has shifted to the two-drug regimen instead of the traditional three-drug regimen for treating HIV-positive individuals. Currently, INSTI—dolutegravir (DTG) with NRTI—lamivudine (3TC) and DTG with NNRTI—rilpivirine is the two-drug regimens approved by the Food and Drug Administration (FDA). DTG with 3TC could be used as first-line therapy for HIV-1-positive naive adults. On the contrary, DTG with rilpivirine can be administered to HIV-1-positive adults with HIV-1 RNA load of fewer than 50 copies per mL with at least 6 months of use of ART with no history of treatment failure components as a replacement to their used ART regimen. Both mentioned two-drug regimens are once-daily single tablets, aiming to reduce potential long-term toxicities of drug–drug interactions and protect drugs that are spared for future use. ^6,7

The two-drug regimen—DTG with rilpivirine is used in Japan, the U.S., Europe, and Canada. ^8,9 A two-drug regimen is an innovative approach, and without a doubt, due to its benefits in real life, it will be adapted widely soon in the future. ¹⁰ In addition, in injectable formulation, another two-drug regimen consisting of cabotegravir and rilpivirine has been approved by the FDA as a complete regimen for HIV-infected adults administered once a month. ^8,9

Rilpivirine has been recently used as a component of the ART regimen available in Turkey. However, few studies have analyzed the rilpivirine-associated mutations before being used by patients. Also, the two-drug regimen and injectable formulation have not been initiated in Turkey. We know that E138K/Q has been potentially added to the expanded list of NNRTI SDRMs (the list of surveillance drug resistance mutations) since 2009, as they are associated with reduced susceptibility to rilpivirine, and their prevalence has since increased. ¹¹ On the contrary, a parenteral, long-acting rilpivirine was developed for potential use as a pre-exposure prophylaxis (PrEP) agent and to improve adherence to ART in HIV-infected individuals. ¹² Hence, before their introduction, which includes the rilpivirine, its associated mutations should be monitored before use.

Thus, this study aimed to investigate the E138 mutations to reveal the situation of rilpivirine-associated resistance in Turkey. The Kocaeli University database was analyzed from 2013 to 2021 for HIV-1-positive individuals. For this study, only naive HIV-1-positive individuals were further analyzed to determine the prevalence and dynamics of rilpivirine-associated mutation/resistance at the E138 position in the reverse transcriptase (RT) region of the HIV-1 genome.

Materials and Methods

Results

Initially, 7232 HIV-1-positive individuals were included. In this study, 43 postmortem samples and ART-receiving 791 patients were excluded, and 6398 newly diagnosed and treatment-naive HIV-1-positive individuals were further analyzed. Most patients were young males with a median age of 36.9. Of the 6398 treatment-naive individuals, 424 (6.6%) possessed mutations in the E138 position. Demographic information and clinical presentations of the treatment of HIV-1-positive naive individuals diagnosed in the years between 2013 and 2021 in Turkey are categorized as E138 mutation-negative or -positive and are summarized in Table 1.

Furthermore, the outlook of antiretroviral treatment regimens between 2013 and 2021, according to the Kocaeli University HIV-1 drug resistance database, was given to understand the situation better (Table 2).

Mutations in the E138 position in RT were analyzed. HIV-1-positive naive individuals with and without the E138 mutation indicated no significant difference in gender, age, CD4+ T cell count, and HIV-1 transmission route-heterosexual contact. On the contrary, a substantial difference in the HIV-1 RNA load was higher in the HIV-positive individuals with the E138 mutation than without the E138 mutation (p = .044). Also, the E138 mutation was significantly observed in the B subtype in contrast to the non-B subtypes and the circulating recombinant forms (CRFs) (p < .001) (Table 3).

The majority of the HIV-1-treatment-naive individuals with the E138 mutation (n = 424, 100%) were subtype B (n = 168, 40%), followed by the CRFs (n = 115, 33.1%) and non-B subtypes (n = 141, 26.9%). Most of the E138 mutations were E138A (n = 338, 80%) followed by E138G (n = 70, 16.5%) (Table 4).

Table 5 indicates that the single and combined E138 mutation compared in E138 defined patients (n = 424) according to the HIV-1 subtype B, nonsubtype B, and CRFs. There was no significant difference between the HIV-1-positive individuals carrying substitutions at codon 138 in RT on NNRTI and the additional mutations (p = .359).

Discussion

Rilpivirine, a new NNRTI of the diarylpyrimidine family inhibitors used in managing HIV-1 infection, has recently been introduced to Turkey to treat HIV-1-positive patients. However, this region's comprehensive rilpivirine-associated resistance mutations with an extensive database have never been evaluated previously. Thus, this study has investigated the 6398 HIV-1 favorable treatment-naive individuals and revealed that 424 (6.6%) of the primary mutations in the E138 position in the HIV-1 genome are associated with the resistance development of rilpivirine. According to the TREAT Asia HIV-1 Network, among 1627 HIV-1-naive patients, 48 (3.0%) had at least one or more rilpivirine resistance-associated mutations, with E138A (1%) and E138G (0.7%) being detected, and E138K (0.1%) accompanies these mutations. ¹⁴

HIV-1 subtype A is predominant in Russia, and E138A polymorphic mutations are detected in ∼4%–8%. ¹⁵ Parczewski et al's findings in Northern Poland show that the prevalence of E138A and E138G mutations is 3.7% and 0.8%, respectively. ¹⁶ According to the systematic review by Calvez et al, they determined the prevalence of rilpivirine resistance-associated mutations in HIV-1-naive individuals, E138A/G/K/Q/R was identified as 0.7%. ¹⁷ Detection of rilpivirine-associated E138 mutations may vary by geographic region and HIV-1 subtype. Therefore, surveillance of rilpivirine-associated mutations may be necessary, which may have an essential role in initiating a rilpivirine-containing ART regimen.

Although the HIV transmission route trends were similar in the E138 negative and positive groups, fewer coinfection patterns and comorbidities were seen in the E138-positive group (Table 1). We did not find it necessary to consider these observations with statistical significance. We aimed to get to know the patient population based on the study. Our findings show that HIV-1-positive individuals with or without the E138 mutation indicated no significant difference in gender, age, and CD4+ T cell count categories. However, individuals with the E138 mutation had significantly higher HIV-1 RNA load than individuals without the E138 mutation (p = .044) (Table 3). In addition, E138 mutations were mainly observed in the HIV-1 B subtype in contrast to the non-B subtypes and the CRF (p < .001) (Table 3).

The prevalence of E138 mutations in HIV-1 ART-naive patients was shown to vary by HIV-1 subtype, with the highest for C (6.1%), F (5.1%) and A (3.3%) subtypes. ¹⁷ E138A mutations are detected in 4%–8% of the HIV-1 A6 subtype, common in the Russian Federation. ¹⁸ Our results are understandable. Subtype B is the predominant pandemic HIV-1 strain in Turkey. However, studies conducted in the last decade show that the prevalence of A1 has an increasing trend and is between 0.9% and 16%. ^13,19,20 The findings suggest that in treatment-naive patients, the prevalence of E138 mutation may vary according to the HIV-1 subtype. On the contrary, as HIV-1 viral load increases in patients, the genetic position at the encoding NNRTI target in RT may be more labile, and the incidence of E138 mutation may increase.

It is reported that a mutation in the E138 position alone may decrease the rilpivirine susceptibility two to threefold. Combining E138 with other NRTI-resistance may reduce rilpivirine susceptibility up to fivefold. ^21,22 Of the 393/424 who possessed E138 mutation single, 31/424 harbored E138 mutation with a combination of other mutation(s). There was no significant difference in the HIV-1 subtypes between single and combined E138 mutations in this study (p = .359) (Table 5). On the contrary, combining mutations with NNRTI resistance for rilpivirine were K103N+E138A (five times), K103N+E138Q (two times), V106I+E138A (two times), and V106VI+E138EA (two times) observed (data not shown). The NRTI resistance-associated mutation M184I can be seen in combination with E138K and, to a lesser extent, K101E mutations.

Alone, the M184I does not affect rilpivirine susceptibility; however, when combined with E138K or K101E, a 7-fold and 4.5-fold decline in rilpivirine exposure can be observed, respectively. ²³ The M184I mutation was not associated with the combined rilpivirine-associated mutations within this study. Of the 168 subtype B, 150 harbored E138A substitution mutation. On the contrary, the 141 nonsubtype B had 50, and the 141 CRF had 138 E138A mutations. Thus, for Turkey, E138A mutation was in a higher prevalence when compared to other substitution mutations in a total of 338 (80%) (Table 4). Wensing et al have suggested that, most notably, E138A exists in the non-B subtype of HIV-1. ²³

On the contrary, another study has revealed that E138A substitution in HIV-1 RT was more frequent in subtype C. In Sluis-Cremer et al, E138A was present in 350/17481 (2.0%) subtype B sequences and 415/6795 (6.1%) subtype C sequences from RT inhibitors naive HIV-1-infected individuals. ²⁴ For Turkey, the numbers highlighted that the prevalence of E138A mutations was majorly in subtype B, followed by the CRFs. The fact that we have determined the prevalence and types of mutation patterns associated with rilpivirine can provide baseline data and evaluate possible future changes.

In this study, we focused on rilpivirine-associated E138 mutations and accompanying dynamics. To date, 22 rilpivirine resistance-associated mutations have been identified. The significant mutations include E138A/G/K/Q/R/S in the RT region of the HIV-1 genome, which may occur as natural polymorphisms. ^21,23 In their work published recently, Stanford and the French HIV Resistance databases identify that E138A mutations differ in the level of resistance to rilpivirine. ¹⁵ Adopting the two-drug regimens and the injection formulation applied once a month may be vitally essential to HIV-positive individuals, meaning that they will need to take fewer drugs while having the same efficacy outcomes as the traditional three-drug regimen.

Therefore, shortly, intramuscular antiretroviral administrations with cabotegravir may also expand the use of rilpivirine. Our findings may provide valuable insights ahead of possible expansion of rilpivirine use.

Unquestionably, introduction and access to ART are significant in preventing further spreading and managing HIV infection. However, the development of drug resistance mutations reduces therapeutic success drastically. Our results indicate that the E138 positioned mutation in the HIV-1 RT domain, mainly in subtype B, is in the circulation in naive HIV-1-infected individuals; hence, before administration of regimens consisting of rilpivirine, resistance monitoring is highly recommended in effective patient management.