Virological failure in drug-naïve HIV-1-infected patients treated with lopinavir/ritonavir-based regimens has rarely been associated with selection of resistance mutations at the protease gene, and mainly in individuals with low drug compliance . Selection of the protease change I47A has been the most frequent mutation found in this situation [1,2], although other substitutions, including V32I, have also been associated with high-level lopinavir resistance in prior drug-naïve individuals [1,2]. Mutation I47A may cause a more than 100-fold loss of susceptibility to lopinavir [2,3]. Interestingly, HIV-2 may be prone to select for 47A because this virus only requires a single nucleotide substitution for a shift to arginine (GCA) at this position. Although HIV-2 naturally has a valine (GTA) at codon 47 , the wild-type HIV-1 has isoleucine at this position encoded by AUA, and therefore two nucleotide substitutions are required for the occurrence of I47A.
At the last Drug Resistance Workshop held in Barbados in June 2007, Nikhuis et al.  reported three HIV-1 drug-naïve individuals who failed a first-line regimen including lopinavir/ritonavir, all of whom selected a L76V change at the viral protease that apparently was responsible for lopinavir resistance. In all instances, L76V was accompanied by the M46I substitution. Site-directed mutagenesis experiments demonstrated that the single L76V mutation significantly reduced lopinavir susceptibility (by 12-fold on average), although with a relatively high cost in diminished replication capacity, which was compensated by the accumulation of M46I that did not confer any lopinavir resistance on its own . Interestingly, L76V caused cross-resistance to amprenavir (five-fold loss of susceptibility) and, conversely, produced hypersusceptibility to atazanavir, saquinavir and tipranavir.
Given that lopinavir/ritonavir is one of the most widely used protease inhibitors , we examined the prevalence of mutation L76V in a relatively large clinical database of resistance genotypes derived from antiretroviral-experienced patients in Spain. From a total of 4457 genotypes from HIV-1-infected individuals tested since January 1999 to June 2007, 1376 belonged to patients failing protease inhibitors. Overall, 37 patients harboured viruses with the protease change L76V (prevalence of 2.7%). Of note, 26 of them (70%) were taken lopinavir/ritonavir at the time of failure, whereas the remainder were under other distinct protease inhibitors. Focusing exclusively on genotypes belonging to patients failing lopinavir/ritonavir, L76V was found in 26 out of 510 specimens (5.1%). Figure 1 shows the prevalence of mutations so far reported to be associated with significant lopinavir resistance . Interestingly, the only change that was significantly more prevalent in patients failing lopinavir compared to patients failing other protease inhibitors was L76V (5.1% versus 2.7%, P < 0.01).
It is noteworthy that 33 out of the 37 patients harboring L76V viruses had codon 46 changes (46I in thirty and 46L in three), which are well known protease inhibitor resistance substitutions . However, other primary resistance mutations at the protease were seen in almost all these patients, including I54L/M/V (n = 27), L90M (n = 22), V82A/F/S/T (n = 17), I84V (n = 15) and I47V (n = 1). Moreover, all patients with L76V viruses had previously failed other protease inhibitors before the current failure on lopinavir. When the estimated impact of L76V on lopinavir resistance was assessed using the Stanford drug resistance rules in our series of patients, the presence of L76V only contributed to slightly increase lopinavir resistance because the accompanying mutations already accounted for intermediate or high-level resistance to the drug in all cases. It should be noted that all patients with L76V identified in our database were infected by HIV-1 subtype B strains, despite 9% of the whole database study population carrying non-B subtypes.
None of the subjects carrying L76V viruses harboured I47A, which was present in only four patients in the whole study population, suggesting that I47A and L76V may represent divergent pathways for lopinavir resistance, a hypothesis which is supported by structural models . However, our results suggest that only I47A may be selected in the absence of other protease inhibitor resistance mutations and be responsible for clinically relevant lopinavir resistance. By contrast, selection of L76V generally occurs in patients who already have failed other protease inhibitors and have accumulated other resistance mutations at the protease.
Our study, however, should be interpreted with caution. In all studies conducted so far in which L76V has been recognised in patients failing lopinavir, the drug was taken as monotherapy, and this strategy has never been used in our institution. As the selection of resistance mutations may differ in patients under mono- or combination therapy, our results may only apply to those patients taking lopinavir along with at least another two antiretroviral drugs. It may be the case that the minimal impact on lopinavir susceptibility caused by L76V may explain its selection in those patients only treated with lopinavir monotherapy in whom low exposure to the drug may occur in some body compartments.
Overall, our results suggest that although drugs such as atazanavir and saquinavir may select for unique distinct protease resistance changes, mutations at codons 32, 47 and now L76V appear to be shared in the resistance pathways for fosamprenavir, lopinavir and darunavir [9–11]. Therefore, it may be worth exploring the best way to sequence protease inhibitors in order to minimise the impact of cross-resistance mutations within this family.
This work was supported in part by grants from Fundación Investigación y Educación en SIDA (IES), Red de Investigación en SIDA (RIS, ISCIII-RETIC RD06), Fondo de Investigaciones Sanitarias (FIS) Project CP06/00284 and Project PI06/1826 and Agencia Laín Entralgo.
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