Tipranavir (TPV) and darunavir (DRV) are the latest approved protease inhibitors (PI) for the treatment of HIV infection. Both show potent antiviral activity against most PI-resistant viruses. The RESIST and POWER trials have demonstrated significant greater reductions in plasma HIV RNA and increases in CD4 cell counts using TPV or DRV, respectively, over controls using other ritonavir-boosted PI in patients with extensive PI resistance [1,2]. There are few data, however, about relative differences in antiviral efficacy between TPV and DRV across distinct HIV subtypes .
Twenty-one mutations located at 16 protease positions have been associated with a reduced response to TPV in the RESIST 1 and 2 trials conducted in PI-experienced patients . Loss of optimal response to TPV was associated with the presence of more than eight mutations from this list. The latest International AIDS Society (IAS)-USA panel also has added L90M to the list . More recently, French researchers have reexamined the data from the RESIST studies and proposed an alternative algorithm for TPV resistance in which only six mutations seem to be the main drivers for loss of TPV susceptibility . Finally, based on phenotypic results, Parkin et al.  have been able to identify the changes with the highest (I47V, I54A, V82T) and lowest (I13V, K20R, M36I, M46L) impact on TPV susceptibility.
Information on DRV resistance is still scarce and mainly derived from the POWER studies. In these trials, 11 mutations at the protease positions were specifically associated with DRV resistance  and this list has been assumed by the IAS-USA panel . When three or more of these changes are present, DRV activity is thought to be reduced .
The natural genetic variability in the gene for protease of non-B HIV-1 subtypes results in amino acid polymorphisms that may influence PI susceptibility before exposure to these agents [9,10]. In spite of this variability, proteases from non-B subtypes are generally fully susceptible to the inhibitory activity of most PI drugs [11,12], although a shorter time to develop resistance and/or differential resistance pathways have been reported for some. This is the case for a more rapid selection of V47A in HIV-2 than HIV-1 following exposure to lopinavir  or a preferential selection of L90M instead of D30N in patients treated with nelfinavir and infected with subtypes C and G [14,15]. The aim of this study was to assess the prevalence of TPV and DRV resistance-associated mutations in clinical specimens collected from drug-naive and PI-experienced patients infected with distinct HIV-1 subtypes, and assess their impact on TPV and DRV susceptibility.
All HIV drug-resistance tests performed between January 1999 and December 2006 in a reference HIV laboratory located in Madrid, Spain, were retrospectively examined. Genotypes were split into two categories, as belonging to drug-naive or PI-experienced patients. Genotypes from patients who had failed only reverse transcriptase inhibitors, either nucleoside or nonnucleoside analogues, were excluded from this analysis. Prior treatment exposure and drug regimens at the time of failure were recorded in a case report form. All samples collected from patients who had been exposed to TPV and/or DRV were excluded from this analysis.
Drug resistance testing and HIV-1 subtyping
Genetic sequences from the genes for both HIV protease and reverse transcriptase were obtained from plasma using the Viroseq HIV-1 kit (Abbott Laboratories, North Chicago, Illinois, USA) and an automatic sequencer (ABI Prism 3100; Celera Diagnostics, Alameda, California, USA).
Drug resistance mutations within pol were interpreted following the latest IAS-USA panel list (www.iasusa.org), updated in September 2007 . The following 11 protease resistance mutations were considered for DRV: V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, L76V, I84V and L89V . For TPV, three different resistance mutations lists were assessed. First, the one proposed by Baxter et al. , in which the following 21 changes are considered: 10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D and 84V. The IAS-USA panel added L90M to this list . Finally, a recent French algorithm has reported the following changes as determinants of TPV virological response: 35D/G/K/N, 36I/L/V, 53L/W/Y, 58E, 61D/E/G/H/N/R, 69I/K/N/Q/R/Y and 89I/M/R/T/V . HIV-1 variants were subtyped by phylogenetic analyses, as previously described .
The PhenoSense assay (Monogram Biosciences, San Francisco, California, USA)  was used to determine the susceptibility to TPV and DRV. The results obtained were interpreted taking into account the clinical cut-offs previously established for TPV  and DRV . The lower and upper clinical cut-offs ranged from 2 to 10 for TPV, and from 10 to 90 for DRV. Finally, the replication capacity of recombinant viruses was examined as previously reported elsewhere .
All data are reported as absolute numbers and percentages; as well as mean ± SD. Comparisons were made using the Student's t test for continuous variables, and the Pearson, χ2 or the Fisher's exact tests for categorical variables. Univariate and multivariate linear logistic regression analyses were performed to asses which factors were related to the presence of TPV-resistance mutations. Statistical significance was assumed for P values <0.05. All statistical analyses were performed using the SPSS version 11.0 (SPSS, Chicago, Illinois, USA).
A total of 1364 genotypes were examined, including 1178 belonging to individuals infected with subtype B and 186 with non-B subtypes. In subtype B, 893 genotypes were from PI-failing patients and 285 from drug-naive subjects. For non-B subtypes, the distribution was as follows: 4A, 11C, 12CRF01_AE, 72CRF02_AG, 1CRF06, 1CRF07_BC, 5CRF11, 17CRF12_BF, 5CRF14_BG, 12D, 10F, 34G and 2H. Overall, 49 non-B subtypes were from PI-experienced patients and 137 from drug-naive individuals.
The mean number of prior PI drugs used was higher in patients infected with clade B than non-B subtypes (2.5 ± 1.4 versus 1.2 ± 1.1; P < 0.001). This observation most likely explains why the mean number of PI resistance-associated mutations was higher in the former compared with the latter group (4.9 ± 3.7 versus 4.2 ± 1.8; P < 0.001) using the IAS-USA panel list. The overall mean number of DRV resistance-associated mutations was 0.4 ± 0.9 in clade B and 0.06 ± 0.3 in non-B subtypes (P < 0.001). In contrast, the mean number of TPV resistance-associated mutations was significantly higher in non-B than B subtypes, using the Baxter list (2.7 ± 1 versus 1.2 ± 1.6), the IAS-USA list (2.8 ± 1.03 versus 1.6 ± 1.8) or the French algorithm (3.1 ± 0.9 versus 0.7 ± 0.9). Interestingly, this difference for TPV persisted even when considering drug-naive or PI-experienced patients separately.
In drug-naive patients, some TPV resistance-associated mutations were significantly more prevalent in non-B than B subtypes: I10V (10.9% versus 4.6%), I13V (74.5% versus 23.5%), K20M/R/V (13.1% versus 2.8%), E35D/G/K/N (48.9% versus 28.1%), M36I/L/V (97.1% versus 13.3%), I54M/A/V (4.4% versus 0.7%), G61D/E/G/H/N (13.1% versus 6.3%), H69K (77.4% versus 0.7%) and L89I/M/R/T/V (82.5% versus 1.4%) (Fig. 1). It should be noted, however, that these results were mainly driven by the high proportion of recombinant CRF02_AG viruses in our study population, which represented 39% of all non-B subtypes. By contrast, DRV resistance-associated mutations were rarely seen in the study population and did not show remarkable differences between B and non-B subtypes.
In order to determine the real impact of the presence of TPV resistance-associated mutations seen as natural polymorphisms in the study population, the susceptibilities to TPV and DRV were examined in 29 samples drawn from drug-naive patients. They were infected with the following subtypes: 1A, 3C, 2CRF01_AE, 9CRF02_AG, 1CRF12_BF, 3CRF14_BG, 3F and 7G. Phenotypic results are given in Table 1. All subtypes showed full susceptibility to DRV and 93% to TPV. Interestingly, two specimens showed reduced TPV susceptibility. Both belonged to subjects infected with subtype F, and fold-changes were 2.7 and 2.1, respectively. TPV-resistance associated mutations in these specimens were K20R, E35D and M36I in both cases, in addition to L89M in one of them. In this last patient, the addition of mutation L89M seemed to confer a fold-change of 2.7 to TPV. Determining the specific contribution of each of these mutations on TPV susceptibility in clade F would require testing a larger number of subtype F specimens with paired genotypic and phenotypic results. Interestingly, subtype F specimens were also the ones with the highest replication capacity among the HIV-1 subtypes tested (Table 1).
This study shows that all HIV-1 subtypes examined from drug-naive HIV-1 individuals showed full susceptibility to DRV and 93% to TPV. Interestingly, two specimens showed reduced TPV susceptibility. Both belonged to subjects infected with subtype F, and fold-changes were 2.7 and 2.1, respectively. TPV-resistance associated mutations in these two specimens were K20R, E35D and M36I, with L89M also in one of them. In the Stanford HIV database , in which 182 PI-naive individuals infected with clade F viruses are recorded, mutations K20R, E35D, M36I and L89M are relatively common (prevalence of 34%, 94%, 95% and 69%, respectively). Since these changes are similarly seen in other HIV subtypes with no recognizable impact on TPV susceptibility, it must be the protease genetic background in subtype F that accounts for this particular effect on TPV susceptibility. Furthermore, our observations are in agreement with a prior report from Abecasis et al. , who suggested that subtype F viruses could be less susceptible to TPV than subtypes C, CRF02_AG and G.
It is noteworthy that subtype F showed the highest replication capacity among the HIV-1 subtypes tested. Hypothetically, the presence of some minor TPV resistance-associated mutations in subtype F could compensate for the reduced fitness caused by the selection of major TPV resistance-associated mutations. In other subtypes, a low replication capacity was generally recognized in the presence of some minor TPV resistance-associated mutations.
Drug-naive individuals infected with non-B subtypes were found to harbour a relatively high number of TPV resistance-associated mutations, but only samples containing subtype F showed reduced TPV susceptibility. Based on this observation, it might be worth performing HIV subtyping before considering TPV in salvage therapy in countries where subtype F is prevalent, such as Brazil, Argentina and Uruguay [22,23]. Whether the presence of some minor TPV resistance-associated mutations could facilitate the development of major TPV resistance mutations in non-B subtypes needs to be further examined. A shorter time to develop a significant loss of susceptibility to TPV has recently been reported for HIV-2 isolates, which show 10V, 36I and 47V as natural polymorphisms .
Given that our results are based on the TPV score derived from studies conducted in subtype B isolates, and the current TPV algorithm could be inadequate for non-B subtypes, future TPV scores should be developed including values derived from non-B viruses, ideally confined to nonpolymorphic positions .
In summary, the analysis of a relatively large number of HIV-1 genotypes derived from clinical specimens showed that TPV resistance-associated mutations were relatively prevalent among drug-naive individuals infected with non-B subtypes. This is not the case for DRV, where specific resistance mutations were largely dependent only on extensive prior PI exposure. Our results show that while HIV clade has no influence on DRV susceptibility, some F subtypes might have reduced TPV susceptibility. This differential impact of HIV-1 subtypes on TPV and DRV susceptibility might favour the selection of one drug over the other in the design of salvage regimens in PI-experienced patients.
We would like to thank Jonathan Schapiro and Robert Shafer for helpful comments.
Sponsorship: This work was funded in part by grants from Fundación Investigación y Educación en SIDA (IES), Red de Investigación en SIDA (RIS, ISCIII-RETIC RD06) and Agencia Laín Entralgo.
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