To ascertain whether the rapid development of K65R in subtype C viruses may be caused by virulence or replicative fitness, viral isolates were also selected for resistance to lamivudine and didanosine in parallel to tenofovir. No significant differences in times to the development of resistance to lamivudine were observed for all isolates tested, and M184I/V emerged by weeks 8–14 (Table 1). Of note is the fact that resistance to didanosine developed with two subtype C selections with the first emergence of M184V and L74V between weeks 22 and 25 (Table 1). No resistance-conferring mutations were detected by weeks 37–40 in the presence of didanosine with the remaining subtype B and C isolates (Table 1).
The levels of RT activity in amplified K65R viral stocks grown in the absence of drug were compared with matched wild-type controls. The replicative capacities (mean ± SEM) of the four K65R-containing subtype C isolates and the two K65R-containing subtype CRF1_AE isolates, compared with matched wild-type controls, were 62 ± 7 and 51 ± 3%, respectively, compared with 46 ± 5% for the K65R-containing recombinant B clone compared with wild type.
Phenotypic drug susceptibility testing was performed to ascertain the drug sensitivity and resistance profiles of different viral subtypes. The baseline IC50 values for tenofovir sensitivity of the four subtype C isolates was 0.48 ± 0.18 μmol, within a similar range, 0.25 ± 0.15 μmol was observed for subtype B infections. Subtype C and CRF1_AE viruses harbouring K65R were assayed for their relative susceptibility to tenofovir and nucleoside analogues. As shown in Fig. 2, all variants harbouring K65R showed similar 6.5 to 10-fold resistance to tenofovir. These viruses also showed 8–22 ×, 10–23 ×, and 3–16 × levels of resistance to abacavir, lamivudine and didanosine, respectively. These viruses all remained susceptible to zidovudine.
Tenofovir has been proposed as a drug of choice in antiviral drug strategies directed against the global pandemic , as a result of its favourable resistance profile, long intracellular half-life, and marginal adverse effects [17–20]. Current guidelines recommend tenofovir/emtricitavudine (Truvada) or zidovudine/lamivudine (Combivir) with efavirenz as two first-line treatments of choice for antiretroviral-naive patients . Clinical trials to evaluate the efficiency of tenofovir in pre-exposure prophylaxis have been initiated in Africa, Asia, and the United States . Among the reasons for choosing tenofovir over such drugs as lamivudine has been the reduced risk of developing resistance to tenofovir via the K65R mutation.
We have now demonstrated that tenofovir can rapidly select K65R in subtype C viruses in cell culture. The appearance of K65R with tenofovir is as rapid as that of M184V with lamivudine. These findings indicate that tenofovir-based regimens in subtype C infections will need to be carefully monitored. A recent report suggested that K65R is less prevalent in patients harbouring subtype A than in subtype B and C infections . On the basis of our findings, the reduced prevalence of K65R in subtype A may partly be explained by a higher rate of selection in subtype C infections.
The mechanisms involved in the more rapid selection of K65R among subtype C viruses may relate to the sequences that naturally encode a wild-type KKK motif at codons 64, 65 and 66. Only a single point mutation is required for the emergence of K65R in each of the viral subtypes evaluated. Conceivably, the nucleotide environment of the position 65 codon plays a role in governing the frequency of K65R substitutions, allowing for the more rapid amplification of K65R viruses under conditions of drug pressure. It does not appear that K65R subtype C variants are less impacted in terms of replication capacity, compared with wild-type, than subtype B viruses containing this same substitution .
There are potential limitations in extrapolating in-vitro selection data to the in-vivo situation. Site-directed mutagenesis studies in which the AAA codon 65 of a subtype B clone is replaced by AAG and in which the AAG at codon 65 in subtype C viruses is replaced by AAA are ongoing. Previous work showing that a V106M substitution in subtype C viruses is selected by efavirenz provides proof of concept that in-vitro findings may be reflected in the clinical setting . Several recent studies have shown non-responsiveness to tenofovir/lamivudine/abacavir regimens, leading to the rapid appearance of K65R in 48–68% of patients [24,25]. In contrast, the 934 study showed clinical benefit of a tenofovir/emtricitabine/efavirenz regimen with no appearance of K65R . Such studies demonstrated that drug potency, low genetic barrier to tenofovir resistance, and physiological factors may all contribute to differential responsiveness to tenofovir . Our study highlights the need for careful evaluation of tenofovir-based regimens in subtype C infections.
Although the K65R mutation remains relatively uncommon, an increase in its prevalence in western countries has been observed over the past 4 years, as a result of the increased clinical use of tenofovir [26,28–30]. The K65R mutation may also be associated with the Q151M resistance pathway . Our group has shown with others that three out of eight treated patients in Botswana developed K65R with didanosine-containing regimens . Of note is the fact that the the four subtype C isolates selected with didanosine in this study did not develop K65R, although M184V and L74V were observed in two selections.
It is still unknown whether K65R will become increasingly prevalent in subtype C infections after the usage of tenofovir. Our findings indicate the need for vigilance in adapting antiretroviral regimens and prophylactic schedules for use in developing countries, and that subtype differences in regard to the development of drug resistance need to be carefully monitored whenever antiretroviral drugs are introduced.
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