M184V is associated with a low incidence of thymidine analogue mutations and low phenotypic resistance to zidovudine and stavudine

The resistance of HIV clinical isolates with or without M184V was analysed in relation to plasma HIV-1-RNA level and time on therapy. The number of thymidine analogue mutations (TAMs) was lower in isolates with M184V, this was independent of plasma HIV-1-RNA level and time on therapy for T215F/Y, D67N and L210W. This suggests a direct effect of M184V on the reduced selection of TAMs. Lamivudine use was significantly associated with lower median fold resistance to zidovudine and stavudine. Treatments with lamivudine or lamivudine/zidovudine retain significant activity despite selection of the M184V [1,2]. Larder et al. [3] showed that lamivudine and M184V delay the development of mutations and resistance to zidovudine. M184V virus is more susceptible to zidovudine than wild type in the absence of thymidine analogue mutations (TAMs) [4], and regains susceptibility to zidovudine in the presence of TAMs [5]. TAMs confer resistance to zidovudine and stavudine by enhancing the removal of incorporated zidovudine or stavudine via pyrophosphorolysis, whereas M184V decreases pyrophosphorolysis reducing TAMs resistance [6,7]. This may explain the reversal of zidovudine resistance, and the delay in the selection of TAMs by M184V [6,7]. M184V virus is more susceptible to tenofovir [8] compared with wild-type virus, and results in increased virological efficacy. Other features of viruses with M184V include reduced fitness compared with wild type [8]. We carried out a retrospective analysis of the association between M184V and the number of TAMs and phenotypic fold resistance to nucleoside reverse transcriptase inhibitors. Baseline plasma samples from all patients in the CNAB3002 study were obtained, patients’ treatment history and cumulative time on antiretroviral therapy (ART) were known, and patients were stratified for previous/current lamivudine use [9]. Genotypes were obtained by HIV-1 reverse transcriptase population sequencing using ABI377 [9], and phenotypes were carried out at Virco NV (Antivirogram, Mechelen, Belgium) [10]. Univariate analyses included ranked non-parametric test (Mann–Whitney Wilcoxon) for the phenotypic fold resistance used as continuous variables and Fisher's exact test for the comparison of genotypes and percentage greater than fourfold resistance. Multivariate analyses consisted of logistic regression models on the frequency of TAMs using the baseline HIV-1-RNA level and time on therapy as covariates. All statistical tests were two-sided and used a 5% alpha level. For the genotypic and phenotypic patient populations, the baseline HIV-1-RNA level (3.7 log10 copies/ml) and time on therapy (60 weeks) were similar between lamivudine-experienced and naive patients. Overall, 72% of patients had lamivudine in their regimen; 76% had dual, 18% triple, and 6% quadruple therapy. Zidovudine and stavudine were used at similar frequencies in the lamivudine experienced (74 and 26%) and lamivudine-naive patients (80 and 20%), respectively. M184V was detected in 99% of lamivudine-experienced patients, but in none of the lamivudine-naive patients (P < 0.001) despite the use of didanosine in 26 out of 39 patients (67%) (Fig. 1). The Q151M pattern was only observed in lamivudine-naive patients (8%, P = 0.02). Wild-type viruses were more common in lamivudine-naive than -experienced patients (13 and 1%, respectively, P = 0.006). A significantly lower incidence of isolates with three or more TAM was observed in lamivudine-experienced than -naive patients (9 and 36%, respectively, P = < 0.001). A significantly higher proportion of viruses from the lamivudine-naive compared with the lamivudine- experienced patients contained D67N (33 versus 8%, P < 0.005), K70R (33 versus 16%, P < 0.005), L210W (26 versus 7%, P < 0.005) and T215Y/F (56 versus 27%, P < 0.005).Fig. 1.: Frequency of genotypic patterns for isolates from lamivudine-experienced and -naive patients. All isolates from lamivudine-experienced patients also contained M184V. ░ Lamivudine experienced (n = 101); ▪ lamivudine naive (n = 39).In a multivariate analysis, the incidence of TAM was associated with the plasma HIV-1-RNA level, time on therapy, M184V, or a combination of these factors (Table 1). A higher plasma HIV-1-RNA level was associated with an increased frequency of D67N, K70R and T215Y/F; a longer time on therapy was associated with an increased incidence of M41L, D67N, L210W, and T215F/Y. After adjustment for treatment duration and plasma HIV-1-RNA level, M184V remained significantly associated with lower incidences of D67N, L210W and T215Y/F.Table 1: Logistic regression model between incidence of thymidine analogue mutations as outcome variable and baseline covariates including plasma HIV-1-RNA levels at time of genotyping, cumulative time on therapy and presence of M184V mutation. Phenotypic data were obtained for 64 lamivudine-experienced and 20 lamivudine-naive patients. M184V isolates had a significantly lower median fold resistance for zidovudine (1.8 versus 26) and stavudine (1.1 versus 2.1) (Table 2). This stavudine result is similar to the 2.2-fold decrease reported by Picard et al. [11] in patients failing stavudine/didanosine, which suggests that this low fold increase in the 50% inhibitory concentration (IC50) for stavudine is of clinical relevance. M184V was also associated with a higher fold resistance for abacavir (2.3 versus 1.7) and zalcitabine (1.6 versus 1.0), but most isolates remained susceptible to abacavir and zalcitabine (83 and 95% with less than fourfold resistance, respectively). M184V was associated with a lower frequency of resistance to zidovudine, 36 versus 90% greater than fourfold resistance in the lamivudine-experienced versus -naive patients (P < 0.0001). There was a trend for M184V to be associated with a lower frequency of resistance to stavudine at the four-fold cut-off, although it is known that a lower cut-off of 1.7 fold is more clinically relevant (8 versus 20% greater than fourfold resistance in lamivudine-experienced versus -naive). In the multivariate analysis, M184V remained associated with a decreased incidence of zidovudine resistance (P < 0.0001), an increased plasma HIV-1-RNA level remained associated with an increased incidence of zidovudine resistance (P = 0.03), but time on therapy was not associated with odds of resistance to zidovudine (P = 0.1). The multivariate analysis could only be carried out for zidovudine because of low frequencies of fold resistance greater than four for all other drugs.Table 2: Median fold resistance to nucleoside reverse trancriptase inhibitors for isolates from lamivudine-experience and -naive patients. This study showed that time on therapy was associated with a higher incidence of all TAMs, the plasma HIV-1-RNA level with a higher incidence of D67N, K70R and T215F/Y, and that M184V was independently associated with a lower incidence of D67N, L210W and T215Y/F. This suggests that M184V in itself may delay TAMs, consistent with another study comparing stavudine/didanosine to stavudine/lamivudine, in which M184V isolates contained fewer TAMs [12]. In particular, T215Y/F was more common with stavudine/didanosine (11/24, 46%) than stavudine/lamivudine (5/23, 22%) [12]. Moreover, in the ALBI trial, M184V detected only in the zidovudine/lamivudine arm was associated with a lower frequency of TAMs, particularly the T215Y mutation (62% with no lamivudine versus 9.5% with lamivudine) [11]. The association between M184V and fewer TAMs may be caused by the mutation partly reverting the mechanism of zidovudine and stavudine resistance [6,7,13]. Isolates with M184V would require more TAMs than isolates without M184V to achieve similar levels of thymidine analogue resistance and replicative advantage under zidovudine or stavudine selective pressure. Isolates with M184V and without TAMs should have a higher resistance barrier to zidovudine and stavudine, decreasing their chance of selection and outgrowth. The removal of lamivudine, which causes a shift to wild-type viruses at position 184, would result in viruses with a higher propensity to develop TAMs and resistance to zidovudine and stavudine. In conclusion, M184V is significantly associated with a lower incidence of TAMs and a lower incidence of resistance to zidovudine and stavudine. As the majority of patients in this study were on dual therapies, it would be premature to generalize these results to patients receiving first-line highly active antiretroviral therapy (HAART). However, the results suggest that patients on HAART, if maintained on a failing regimen, would have less resistance to thymidine analogues with lamivudine in the regimen than without. HAART should be changed soon after virological failure to avoid the accumulation of mutations, especially in regimens without lamivudine. Prospective studies to investigate the benefit of maintaining selective pressure on M184V after switching to a new regimen are warranted. Mounir Ait-Khaleda Chris Stonea Gillian Amphlettb Bonaventura Clotetc Schlomo Staszewskid Christine Katlamae Margaret Tisdalea on behalf of the CNA3002 International Study Team.