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Genetic variation at NNRTI resistance-associated positions in patients infected with HIV-1 subtype C

Grossman, Zehava; Istomin, Valerya; Averbuch, Dianab; Lorber, Margalitc; Risenberg, Klarisd; Levi, Itzchake; Chowers, Michalf; Burke, Michaelg; Yaacov, Nimrod Barh; Schapiro, Jonathan Mifor the Israel AIDS Multi-Center Study Group

Clinical Science: Concise Communication

Objective: Genetic differences between subtypes of HIV-1, even when not associated with key resistance mutations, are known to affect baseline susceptibility to specific antiretroviral drugs and resistance-development pathways. We studied the prevalence and patterns of non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated mutations in HIV-1 subtype C-infected patients.

Method: We analysed the genetic variation at sites associated with NNRTI and nucleoside reverse transcriptase inhibitor resistance in subtype C- versus B-infected patients, both drug-naive and -experienced. We extended the comparison to subtype B records from the Stanford database.

Results: A total of 150 subtype B and 341 subtype C-infected patients were studied. No significant differences were found in treatment and clinical parameters between the groups. In NNRTI-naive patients, changes in NNRTI positions were present in 9.3% of subtype B- versus 33.1% of subtype C-infected patients (P < 0.001). Differences were seen in both drug-naive (subtype B, 10.0% versus subtype C, 50.1%; P < 0.021) and drug-experienced NNRTI-naive patients (subtype B, 9.0% versus subtype C, 23.8%; P < 0.001). In NNRTI experienced patients, the number of A98G/S changes was significantly higher in subtype C patients treated with either efavirenz or nevirapine (P < 0.0001), and V106M was higher in efavirenz-treated subtype C-infected patients (P < 0.0001). The average mutation rates were 1.26 and 1.67 per patient for subtypes B and C, respectively (P = 0.036). The frequency of nucleoside associated mutations, but not M184V, in treated patients was significantly higher in subgroup B-infected patients (P = 0.028).

Conclusion: Collectively, these data indicate that genetic variation at NNRTI resistance-associated positions such as V106M and A98S is substantially greater in subtype C-infected patients than in subtype B-infected patients. The natural structure of each subtype probably affects the frequency and pattern of drug resistance mutations selected under treatment.

From the National HIV Reference Center, Central Virology Lab, Public Health Laboratories, Ministry of Health, aHillel Jaffe Medical Center, Hadera,bHadassah University Hospital, Jerusalem, cRambam Medical Center, Haifa, dSoroka Medical Center and Ben Gurion University, Beer Sheva, eSheba Medical Center, Tel-Hashomer and Tel Aviv University Sackler Medical School, Tel Aviv, fMeir Medical Center, Kfar-Saba, gTel Aviv Sourasky Medical Center and Tel Aviv University Sackler Medical School, Tel Aviv, and the hDepartment of Computer-Science, School of Mathematics, Tel Aviv University, Tel Aviv, Israel. *See Appendix for additional coauthors.

Correspondence to Z. Grossman, National HIV Reference Center, Central Virology Lab, Sheba Medical Center, Tel Hashomer 52621, Israel. E-mail:

Received: 2 February 2003; revised: 6 August 2003; accepted: 8 September 2003.

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Subtype B HIV-1 is the predominant subtype in the developed countries, but most infected people worldwide carry non-B virus [1,2]. HIV subtypes show characteristic patterns of amino acids expressed at specific positions throughout the viral genome [3–8]. Differences between subtypes exist in the protease and reverse transcriptase (RT) genes [9–16].

Non-nucleoside reverse transcriptase inhibitors (NNRTI) are increasingly used in drug combination therapy [1,17–20]. The evolution of resistance to NNRTI, following treatment with these drugs, has been studied almost exclusively in subtype B-infected individuals [18,21,22]; relatively little is known about NNRTI effects on non-subtype B strains [23–25]. It was shown for the protease gene that differences between subtypes in the baseline polymorphisms may lead to the development of unique patterns of resistance to each drug and to differences in efficacy of particular regimens in the treatment of patients infected with different subtypes [13].

In Israel, subtypes A, B and C are prevalent [26–31], HAART is available to all patients, and genotypic resistance testing is performed to help guide treatment decisions [32,33]. We performed a comparative analysis of genetic variation in the HIV-1 RT genes of subtype B and subtype C, in drug-naive and drug-experienced patients, focusing on the variation at NNRTI resistance-associated positions.

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Clinical specimens and database

Samples were sent to the National HIV Reference Laboratory from HIV treatment centres, along with demographic information, detailed antiretroviral treatment history, current and past CD4 cell counts and HIV RNA levels. All subtype B and C samples submitted between August 1999 and May 2003 are included in this study. Data were compared to the large Stanford public database [10].

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HIV-1 RNA extraction and sequencing

Viral RNA was isolated from patient blood plasma using the QIAamp kit (Qiagen, Hiden, Germany) according to manufacturer's instructions. The RT gene (codons 38–247) was sequenced as described previously [11].

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Statistical analysis

Chi-square test, Fisher's two-tailed exact test and Mann–Whitney test were used in comparing clinical data and mutation frequencies.

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Sample classification

A total of 150 subtype B and 341 subtype C patients were divided (Table 1) into drug-naive (30 subtype B and 87 subtype C), NNRTI-naive (drug treated, but not with NNRTI; 78 subtype B and 164 subtype C), and NNRTI treated (42 subtype B and 90 subtype C). Subtype identification was based on comparing the polymerase sequence with consensus sequences [10].

Table 1

Table 1

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Patients and treatments

Subtype B and subtype C patients were treated by the same physicians and in the same clinics. Overall usage of antiretroviral drugs was similar in the two groups. Efavirenz (EFV) and nevirapine (NVP) were commonly used in combination regimens, usually with nucleoside reverse transcriptase inhibitors (NRTI) as the first or second regimen.

Most drug-naive patients had recently been diagnosed. The majority of subtype B patients were born and infected in Israel, former Soviet Union and various western countries. The birthplace of most subtype C patients or of their parents was Ethiopia (93%); 30% of the naive and 11% of treated subtype C patients were infected in Israel. Demographic data varied between the subtype B and C groups in a number of additional parameters: 82% of subtype B patients were male compared to 47.8% of those infected with subtype C; in the subtype C group, 19% were vertically infected children compared with 3.3% in the subtype B group. The main risk groups for subtype B HIV transmission consisted of men who have sex with men, heterosexuals, injecting drug users and receivers of blood transfusion (39%, 29%, 19% and 8%, respectively), while subtype C transmission was through heterosexual relations (81%) and mother-to-child transfer (19%). The viral load and CD4 cell counts at time of treatment failure were similar for the two groups (Table 1, upper section).

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Mutations and polymorphisms

Frequencies of nucleoside associated mutations (NAM), M184V, and NNRTI associated mutations are shown in Table 1 (lower section) for three groups: drug-naive patients, treated patients who did not receive NNRTI, and those who received NNRTI. Basic polymorphism in naive subtype C virus was found at positions A98G/S, A62V and V179I. These three mutations were found both in drug-naive subtype C patients infected outside Israel, where the population was not subject to drug treatment, and in those infected in Israel. Only in the latter, additional mutations, both NAM and NNRTI associated, were found, illustrating the emerging problem of resistant virus transmission in this population. Altogether, mutations were found in three of 30 drug-naive subtype B-infected patients (0.1 mutations per patient) and in 44 of 87 drug-naive subtype C-infected patients (0.61 mutations per patient; P < 0.021). Of those, 21 patients had only A98S (16), K101R (2) or V179I (3) and 23 had other mutations, with or without A98G/S. Twelve patients had between two and eight mutations associated with two classes of drugs. A high rate of 0.49 mutations per naive subtype C-infected patient is noted even when A98G/S is excluded.

NNRTI associated mutations were also more frequent in treated subtype C-infected patients than in treated subtype B-infected patients. The frequency difference was most prominent in the NNRTI-naive subtype C-infected treated patients (Table 1, lower section). NNRTI resistance-associated mutations (A98G, L100I, K103N, V106M, V108I, Y181C, Y188C, G190A) and polymorphisms (A98S, K101E, V106I, V179I) pre-existed in NNRTI-naive subtype C-infected patients, while in NNRTI-naive subtype B-infected patients only A98G/S and K103N occurred (Table 1, lower section). K103N was the most prevalent mutation in NNRTI-treated patients, 42.9% and 38.9% in subtype B- and subtype C-infected patients, respectively. Average NNRTI mutation rates in the NNRTI treated group were 1.26 and 1.67 per patient in subtype B- and subtype C-infected patients, respectively; P = 0.036. As previously shown [11], the overall frequency of NAM was higher (about twofold) in subtype B-infected patients and M184V frequency was similar. The overall frequency of mutations associated with NNRTI was significantly higher in C than in B patients (P = 0.019).

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EFV versus NVP

Of 90 subtype C-infected patients treated with NNRTI, 47 were treated with EFV and 37 with NVP as the only NNRTI. Similarly, 24 and 10 of 42 subtype B-infected patients were treated with EFV and NVP, respectively. Choice of drugs was made by the physicians. We examined accumulation of mutations under each treatment and, as our NNRTI-treated B cohort was small, compared our subtype B- and subtype C-infected patients to subtype B patients from the large Stanford database (Tables 2 and 3). The most frequent mutation in both subtypes was K103N. Its frequency was significantly lower in subtype C- (44%) than in subtype B-infected patients (62%) following EFV treatment (P = 0.013) but similar in subtype C and B patients following NVP (32% and 38%; P = 0.604). V106M frequency was significantly higher in EFV-treated subtype C-infected patients (24%) than in subtype B patients treated with this drug (0.3%; P < 0.0001). It was found in one subtype C patient treated with NVP. Among the EFV-treated patients 0.1% subtype B- and 4% subtype C-infected EFV-treated patients carried Y188C (P = 0.008). On the other hand, Y188H/L was higher in EFV-treated subtype B patients than in NVP-treated subtype B patients (13% and 4%, respectively; P = 0.008). Y181C was significantly higher in patients treated with NVP in both subtypes (Tables 2 and 3). Even on the background of natural polymorphism, the frequency of A98G/S was higher in NNRTI-treated subtype C patients (31% and 48% in subtype C, and 10% and 14% in subtype B, for EFV and NVP, respectively); in both subtypes, this value almost doubled following NVP treatment. The frequencies of L100I, L101E/L/I, V108I, V179I, G190A and P225H following treatment with either drug were similar in the two subtypes (data not shown). There was no significant difference between the Israeli and Stanford subtype B-infected patients.

Table 2

Table 2

Table 3

Table 3

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The objective of this study was to elucidate patterns evolved under treatment regimens containing NNRTI. To this end, we have comparatively analysed the genetic variation in the RT gene of subtype B and subtype C HIV-1 from drug-naive and drug-experienced patients. We compared the frequency at which individual drug resistance mutations were selected in subtype B and C patients who failed therapy. We further compared our results to a large public-access database.

Patients in our study were not randomized into specific treatment arms. The patients had different ethnic backgrounds, and this may have affected both adherence and pharmacokinetics. Such issues were not studied here. However, patients were treated by the same physicians in the same clinics. The drugs received, follow-up time, baseline viral load and CD4 cell counts were similar (Table 1). Although all the mutations associated with resistance to NNRTI in subtype B-infected patients were found also in subtype C-infected patients, different patterns and frequencies of specific mutations were found in each subtype. Significant differences in frequency of individual mutations were found only for A98G/S, V106M and Y188C. However, the frequency differences for all of the other mutations combined reached significance, with more NNRTI resistance-associated mutations in subtype C patients. This applies to each of the three groups, drug-naive, NNRTI-naive and NNRTI-treated patients. The relatively high frequency of NNRTI resistance-associated mutations in subtype C-infected patients is conspicuous when compared with other RT mutations, the NAM and M184V. As previously reported, the frequency of NAM was higher in B infected patients and that of M184V was equal in subtype B- and subtype C-infected patients. In contrast, NNRTI resistance-associated mutations appear to accumulate rapidly in subtype C patients and to manifest a unique pattern. Even in subtype C patients who were treated with NRTI, and not with NNRTI, there was an increased frequency of mutations associated with resistance to NNRTI. The implied cross-reactivity between NRTI and NNRTI should be further investigated.

Recently, Brenner et al. found that the V106M mutation confers cross-resistance to NNRTI in subtype C virus [24]. This mutation rarely appears in subtype B patients [10] or in subtype C patients treated with NVP [25]. Our results confirm these findings: 24% of our EFV-treated subtype C patients and only one (2.7%) of the subtype C patients treated with NVP developed V106M (P < 0.01). Y188C/H/L was found among patients treated for longer periods (and was not among the early appearing mutations); Y188C frequency was higher in subtype C-infected patients treated with EFV and Y188L was higher in those treated with NVP.

V106M and K103N can coexist: of 13 patients who developed V106M, five (38.5%) also had K103N. The prevalence of K103N following EFV treatment was significantly lower in EFV-treated subtype C patients than in subtype B patients treated with this drug. It seems that in some cases V106M arose in subtype C-infected patients at the expense of K103N. Our findings confirm Brenner's in vitro evidence that EFV selects preferentially for V106M in subtype C-infected patients.

Although not conclusive, these data suggest that baseline polymorphism in the subtype C RT influences the rate at which certain mutations develop in patients failing therapy and the mutation pattern. This may be the case also for other non-B subtypes. In spite of the higher mutation frequency in NNRTI-associated RT positions in subtype C-infected patients, we cannot conclude at this stage that the efficacy of NNRTI in the treatment of subtype C patients is lower in these patients than in subtype B patients (differences in viral load or CD4 cell count were insignificant). Prospective clinical trials and phenotypic assays are needed to address this issue.

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We thank M. Ofir, of VGI, Israel, for excellent technical assistance. M. Amit and Y. Shaked helped in database development. The Stanford HIV database is under the direction of R. Shafer.

Sponsorship: This work was supported by Bristol-Myers Squibb, Israel.

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Additional contributing coauthors

Dan Engelhard, Shlomo Maayan, Hadassah University Hospital, Jerusalem; Zvi Bentwich, Dina Torten, Kaplan Hospital, Hebrew University Hadassah Medical School, Rehovot; Ella Mendelson, Fernando Mileguir, Daniela Ram, Hagit Rudich, National HIV Reference Center, Central Virology Lab, Public Health Laboratories, Ministry of Health; Eduardo Shahar, Einat Kedem, Sholomo Pollack, Rambam Medical Center, Haifa; Bat Sheva Gotessman, Giora Gotessman, Meir Medical Center, Kfar-Saba, Israel.


HIV; subtype B; subtype C; NNRTI; drug resistance; V106M mutation; genetic variation; reverse transcriptase

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