The emerging epidemics in Africa and Asia show increasing genetic diversification, with a shift towards non-B subtypes and circulating recombinant forms (CRF) [1–3]. Effective strategies must be developed to handle this growing wave of non-B infections with particular emphasis on subtypes C and A (including CRF1_AE and CRF2_AG), which presently account for over 50 and 30% of new infections worldwide, respectively [3,4].
Natural polymorphisms in non-B subtypes may influence drug susceptibility, therapy response and emerging pathways to drug resistance [5–10]. HIV-1 subtypes can vary by 10–15% in nucleotides and 5–10% in amino acids in the pol gene encoding reverse transcriptase (RT) and protease . Our laboratory has described a subtype C signature mutation in which a valine polymorphism (GTG) codon at 106 facilitates a V106M transition (GTG→ATG) that confers resistance to non-nucleoside RT inhibitors .
Resistance to tenofovir is extremely difficult to select in culture , although the K65R mutation in RT is associated with such resistance in the clinic. In this study, we describe signature nucleotide polymorphisms in subtype C viruses at RT codons 64, 65, and 66, which may be implicated in emergent resistance to tenofovir. We evaluated the differential times to the development of resistance to tenofovir in cell culture comparing subtype C to other HIV subtypes.
Materials and methods
Viral isolates and selection of drug resistance
Subtype C viral isolates used in this study included BG-05 (AF492600), BG-15 (AF492601), and Mole 18 (AF492607) from Botswana and 4742 (AF492595) from Ethiopia [13,14]. Blood samples harbouring subtype A (n = 1), CRF1_AE (n = 2), CRF2_AG (n = 1), B (n = 4), G (n = 1) viruses were also obtained from treatment-naive patients in Montreal with ethics approval. Viruses were isolated by co-culture of peripheral blood mononuclear cells with cord blood mononuclear cells (CBMC) as previously described [14,15]. The GenBank accession numbers for the Quebec non-B isolates are as follows: 6050 (subtype A-DQ380550), 6343 (CRF1_AE-DQ380549), 6240 (CRF1_AE-DQ385889), 6383 (CRF2_AG-DQ380551), and 5880 (G-DQ380550).
The selection of drug-resistant subtype variants was performed by repeated serial passage of viruses in CBMC in the presence of increasing concentrations of drugs for up to 73 weeks for tenofovir, 30 weeks for lamivudine, and 40 weeks for didanosine. RT assays were performed weekly to assess viral replication . Drug concentrations were escalated over time to maintain effective antiviral pressure, i.e. RT activities (> 10% and < 50%) compared with control cultures grown in the absence of drug. Genotyping was performed at select passages to evaluate time to the development of drug resistance.
Genotypic and phenotypic resistance testing
The sequencing of extracted RNA or complementary DNA was performed by Bayer TruGene or by ABI technology to determine genotypic changes associated with drug resistance . Drug susceptibility was measured in cell culture-based phenotypic assays, determining the extent to which antiretroviral drugs inhibit in-vitro HIV replication. Briefly, CBMC were infected with different subtype viral isolates and the 50% drug inhibitory concentrations (IC50) were ascertained for tenofovir and nucleoside analogues, including zidovudine, lamivudine, abacavir, and didanosine, both pre and post-selection with tenofovir [14,15].
Nucleotide polymorphisms surrounding reverse transcriptase codon 65 in different subtypes
We examined non-B subtype sequences from the Quebec provincial genotyping program to identify natural nucleotide polymorphisms in the HIV-1 RT region that might be implicated in emergent drug resistance. Of note was the fact that subtype C viruses (n = 91/95) show a signature pattern of nucleotide polymorphisms compared with subtype B at RT codons 64 (AAG→AAA), 65 (AAA→AAG), and 66 (AAA→AAG), i.e. a KKK motif. This pattern is specific for subtype C. The codon 64 and codon 65 transitions are absent in subtype B (n = 0/49), A/CRF1_AE (0/35), and CRF2_AG (n = 1/35) infections. The codon 66 polymorphism was, however, observed in 22.4, 37 and 23% of subtype B, A/CRF1_AE and CRF2_AG isolates, respectively. The KKK amino acid motif was common at RT positions 64, 65 and 66 in all subtypes before selection for resistance.
Time to selection of tenofovir resistance in cell culture in different subtypes
To ascertain whether the nucleotide polymorphisms surrounding codon 65 may be implicated in resistance to tenofovir, four representative subtype B and C isolates were passaged in increasing dosages of tenofovir. As shown in Fig. 1, concentrations of tenofovir could be escalated more rapidly in subtype C selections. All four subtype C isolates developed K65R (AAG→AGG) within 12 weeks of serial passage in tenofovir, as rapidly as M184V was selected with lamivudine (Table 1). In contrast, none of the subtype B variants developed K65R in the last time of serial passage, i.e. weeks 34–74 (Table 1). Similarly, none of the subtype A, CRF1_AE, CRF2_AG or G isolates developed K65R at weeks 30–33 of serial passage in tenofovir, although the two CRF1_AE isolates were observed to develop K65R at weeks 55 and 73 (Table 1).
Facilitated development of tenofovir resistance in subtype C viruses
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.
Drug susceptibility of K65R
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.
Sponsorship: This work was sponsored by the Canadian Institutes for Health Research (CIHR), the Canadian Foundation for AIDS research (CANFAR), and Fonds de la Recherche en Santé du Québec-SIDA (FRSQ-SIDA).
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