As with other anti-HIV drugs, HIV can become resistant to protease inhibitors (PIs). This resistance results from mutations in the structure of the protease enzyme.

Mutant strains which have reduced sensitivity to the PIs have a selective advantage in the presence of these drugs. Specifically, the mutations prevent PIs from binding to specific sites on the protease enzyme. Changes can be detected by looking at the genetic code of the protease enzyme.

Unlike resistance to 3TC (lamivudine, Epivir) or non-nucleoside reverse transcriptase inhibitors (NNRTIs), resistance to the PIs usually depends on the accumulation of several mutations, one after the other. The first mutation may be distinctive, associated with one particular protease inhibitor. The first change leads to some loss of sensitivity to the protease inhibitor, but not generally to high level cross-resistance. Nevertheless, this initial mutation damages the virus, making it harder for it to reproduce.

Staging of resistance

Over time, multiple mutations which are common to the other protease inhibitors may develop. These subsequent or secondary mutations are thought to improve the fitness of the virus, leading to a much greater rebound in viral load.

This was demonstrated in a study that found that protease with three mutations was highly drug resistant, but this virus was not very efficient at replicating. A further mutation saw no increase in resistance as measured by the amount of drug needed to stop HIV replication. However, the virus's ability to replicate improved dramatically (Nijhuis 1998).

This discovery is important, because it suggests that the chance of benefiting from a second PI is probably greatest if a switch occurs quickly when resistance is first identified and the virus is unfit and not highly resistant. This confirms the findings of many clinical studies which have shown that people who stay on a failing protease regimen for a long period will have little response to a new PI.

Secondary mutations may also contribute to cross-resistance among the PIs.

Cross-resistance

Cross-resistance means that a person may have HIV that is resistant to a drug they have never taken. Generally, HIV is cross-resistant to several or all of the PIs when primary and secondary mutations have occurred.

Resistance to PIs does not occur in a stepwise or sequential fashion, but as a result of competition between mutants which are present for some time at low levels before circumstances begin to favour one mutant over others, introducing a new dominant mutational pattern (Hance 2002).

Some experts have suggested that it is the interaction of mutations that is crucial in PI resistance, rather than particular mutations or the number of mutations.

Universal protease inhibitor mutations

Mutations at positions 82, 84 and 90 have been identified as single mutations which can cause high level PI cross-resistance. These mutations have been dubbed universal protease inhibitor-associated mutations (UPAMs) or protease-associated resistance mutations (PRAMs).

L33 is considered a UPAM by some experts. A Spanish study which included this mutation in its analysis of response to therapy found that all individuals with two or more UPAMs were fully resistant to ritonavir (Norvir), indinavir (Crixivan), nelfinavir (Viracept), saquinavir (Invirase / Fortovase) and amprenavir (Agenerase; Perez-Elias 2003).

Genotypic tests can be used to identify these universal PI mutations.

Pathways to development of the UPAMs and other PI mutations associated with cross-resistance are described below (Colonno 2002b, 2003; DAquila 2003; Prado 2002; Race 1999):

• Ritonavir, saquinavir and indinavir are associated with primary mutations at 46I/L, 82N/F/T, 84V and L90M which cause PI cross-resistance.

• Nelfinavir is associated with the primary mutations 30N and L90M, and these produce reduced susceptibility to nelfinavir.

• Amprenavir is linked to primary mutations 32I, 50V and 84V causing reduced sensitivity to amprenavir and lopinavir.

• Atazanavir is associated with primary mutations I50L and A71 which produce reduced susceptibility to amprenavir.

• Mutations 54V or 71V/T mutations have been individually associated with PI cross-resistance.

Predicting cross-resistance

Genotypic testing (looking for key mutations associated with resistance) may not be a very reliable method for identifying cross-resistance at the individual level, because genotypic resistance patterns associated with PI resistance are highly variable.

Firstly, specific clusters of PI mutations have been associated with resistance to PIs. For example, mutations 54V with 82A and either 10I or 71V/T have been linked to PI cross-resistance (Race 1999).

Secondly, the total number of mutations on the protease gene have been correlated with the degree of cross-resistance.

Mutations that are associated with greater than tenfold resistance to PIs when present in combinations of two or more include 10, 36, 46, 48, 54, 71, 82, 84 and 90. 43I seems to be a secondary mutation in response to the initial 82 mutation, and is thus associated with improved viral fitness.

Researchers looking at the efficacy of tipranavir in 216 patients who had experienced failure of at least two PI-containing regimens reported that whilst the drug showed activity in people with up to two UPAMs, its effect was poor in people with three UPAMs (Gathe 2003).

Thirdly, the sequencing and combining of anti-HIV drugs may also affect which mutations emerge. Another complicating factor in assessing the potential impact of specific mutations is that HIV can adapt to overcome the effects of these protease mutations. Research has shown that mutations may also develop that can restore the activity of HIV protease despite the presence of resistance mutations in the protease enzyme (Tamiya 2004).

Nevertheless, phenotypic resistance testing has show a high degree of cross-resistance between the PIs. In 75% to 95% of samples, a ten-fold reduction in sensitivity to one protease inhibitor was associated with four-fold or greater cross-resistance to all other protease inhibitors.

The latest data about the resistance patterns seen in PI-treated people and possible cross-resistance can be found at Antiviral Drug Resistance Online (http://www.viral-resistance.com) which provides a database of over 800 drugs and their resistance profiles.

Hypersusceptibility to protease inhibitors

While particular mutations in reverse transcriptase have been associated with improved susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs) and NNRTIs, identification of hypersusceptibility to PIs is a recent development. When virus has a low replication rate, phenotypic tests show that the mutation R41K is associated with hypersusceptibility in all the approved PIs, I98L is associated with hypersusceptibility to amprenavir, indinavir and lopinavir, and I93L improves susceptibility to ritonavir and saquinavir (Martinez-Picado 2005). The N88S mutation has previously been linked to improved sensitivity to amprenavir.

Resistance to nelfinavir

Nelfinavir has a distinctive resistance profile, including the signature mutation D30N, which means resistance to nelfinavir may not cause resistance to the other protease inhibitors. When a regimen containing nelfinavir is not controlling viral replication, the D30N mutation is most likely protease inhibitor-associated mutation to emerge.

One study found that people who ceased to benefit from nelfinavir responded to the ritonavir plus saquinavir combination in 50% of cases. Another study reported that first-line nelfinavir was associated with infrequent cross-resistance when compared with other protease inhibitors. Between 7 and 16% of individuals who failed with nelfinavir were resistant to indinavir, ritonavir or amprenavir, compared with 36 to 56% of those who had failed another PI (Kemper 2001).

Another study found that people who developed the D30N mutation while taking nelfinavir had a significantly better chance of responding well to indinavir than those who developed the L90M mutation. Suppression of viral load to below 400 copies/ml was achieved at week 48 in 56% of patients with the D30N virus versus 18% of patients with the L90M virus (Saah 2003).

In one study, nine of 18 people who stayed on a nelfinavir-containing regimen for six months despite viral rebound developed the D30N mutation, and four had the L90M mutation the signature mutation for saquinavir (Anton 1999).

The D30N mutation has also been associated with improved susceptibility to lopinavir and to amprenavir (Coakley 2002). Hypersusceptibility to amprenavir is likely to result from the nelfinavir-associated mutation N88S, which creates a less fit and less infective strain of HIV (Resch 2002). Mutations at L63P and V77I reduce this hypersusceptibility in the presence of N88S and improve fitness.

Another possible advantage is that nelfinavir resistance makes HIV less fit, slowing down replication. See Viral fitness, drug resistance and the immune system in Anti-HIV therapy: Restoring the immune system.

In people with subtype C HIV-1, the D30N mutation is less likely to develop in response to nelfinavir treatment than in people with subtype B HIV-1 (Grossman 2002), and the virus is more likely to take the L90M resistance pathway.

Despite the unique D30N mutation that develops during exposure to nelfinavir, resistance to other protease inhibitors often means resistance to nelfinavir. In particular, the L90M mutation is associated with cross-resistance to nelfinavir (Dronda 2000).

Resistance to low-dose ritonavir

Ritonavir is now used chiefly as an agent to boost blood levels of other protease inhibitors rather than for any anti-HIV effect of its own. A study of people starting salvage therapy with saquinavir and 100mg of ritonavir found that after 12 months of treatment, 23% with no ritonavir-associated mutations at baseline developed the V82A/F or T mutations (Chaillou 2002).

Resistance to amprenavir

Amprenavir is approved for use in people who have failed previous PI-containing regimens. It may have some activity against HIV which is resistant to indinavir, nelfinavir, saquinavir and ritonavir.

GlaxoSmithKline researchers have identified four primary mutations which cause amprenavir resistance in people who havent previously taken PIs at positions 50, or 54, or 32 plus 47, or less commonly I84V. These primary mutations are often accompanied by secondary mutations, commonly the M46I/L, L10I and L33F mutations (Maguire 2002b; Paulsen 2003). Other researchers have also linked mutations at positions 46, 90 and 93 to amprenavir resistance (Schmidt 2000; Calvez).

Traditionally, the I50V mutation is regarded as the amprenavir signature mutation. It is now known that both the I50V and the I84V mutation confer the greatest level of resistance to amprenavir. However, neither of these mutations causes much cross-resistance to the other protease inhibitors (Maguire 2002b). I50V tends to emerge at higher drug concentrations, and results in a significant loss of fitness. In contrast I54L/M emerges at lower drug concentrations, confers lower level resistance and results in less loss of fitness (Paulsen 2003).

A review of viral isolates from 132 individuals who had failed at least one protease inhibitor showed that 67% were still sensitive to amprenavir, and a further 16% exhibited a four-to-eight fold reduction in sensitivity. Of 62 samples resistant to four PIs, 23 were still sensitive to amprenavir. Prior treatment with indinavir or ritonavir was more strongly associated with amprenavir resistance than prior saquinavir or nelfinavir treatment (Schmidt 2000). However, another study found that people who have virus with the L90M mutation plus secondary mutations may gain little benefit from amprenavir (Klein).

In the case of prior lopinavir treatment, trials are awaited that can provide more information. Lopinavir in PI-experienced patients may select the I84V mutation that causes some loss of response to amprenavir. Whilst both response to both drugs is affected by a change at codon 54, different amino acid changes at this position occur. Whilst lopinavir treatment can lead to a I54V mutant, amprenavir treatment leads to a I54L/M substitution that has not been shown to compromise lopinavir response (Paulsen 2003).

As discussed in more detail below, mutations associated with resistance to amprenavir including the signature mutation at position 50 may cause resistance to ritonavir-boosted lopinavir (Kaletra).

Mutations in the gag gene of HIV have also been linked to amprenavir resistance (Lastere 2003; Maguire 2002).

On a more positive note, the mutation at N88S, associated with resistance to indinavir and nelfinavir, enhances HIV susceptibility to amprenavir. Consequently, amprenavir may be an option for individuals with this resistance mutation, although this theory has not been confirmed in clinical results (Petropoulos 2000).

Resistance to amprenavir appears less likely to emerge during failure of first line therapy if it is boosted with ritonavir. A comparison of the NEAT and SOLO studies showed that whilst patients receiving amprenavir and ritonavir in the SOLO study did not develop amprenavir resistance if their regimen failed, patients in the NEAT study receiving unboosted amprenavir did develop resistance to the drug. However, when compared to nelfinavir recipients in those trials, the rate of acquisition for all drug resistance mutations (including NRTI mutations) was lower in amprenavir-treated patients (MacManus 2003).

Resistance to lopinavir

Abbott's ritonavir-boosted lopinavir (Kaletra) has shown substantial activity against PI-resistant virus and early studies of first-line lopinavir treatment suggested that resistance mutations to lopinavir rarely emerge (Stevens 2003).

However, a range of individual mutations and groups of mutations have been associated with resistance to lopinavir, and resistance to lopinavir is thought to have been under-reported during the early years of availability (Parkin 2003).

The V82A mutation followed by its replacement with V321, M46M/I, and I47A mutations has been associated with high level phenotypic resistance (Friend 2004).

Mutations associated with greater than tenfold reductions in lopinavir susceptibility are 10I, 71V, 90M, 54V, 46I, 84V, 46L, 73S and 20R (Parkin 2003; Harrigan 2001). According to Abbott, manufacturer of lopinavir, virologic failure in the presence of at least three of these mutations was highly unusual in the absence of a change at codon 82 (Calvez 2001). Several studies suggested that people needed at least six or seven lopinavir-related mutations to have a significant loss in susceptibility to lopinavir (Prado 2001; Ruffault 2002). However, subsequent research has shown that high level resistance to lopinavir can occur when only four mutations are present (Prado 2002).

For example, a large number of mutations are not necessary for resistance to lopinavir when the amprenavir-associated I50V mutation is present. Virologic has reported a very high degree of cross-resistance to lopinavir in individuals who had the I50V mutation (mean loss of sensitivity: 130-fold). Another amprenavir-associated mutation conferring very high level resistance to lopinavir has been identified at codon I47A, which evolves from the amprenavir-associated L47V mutation (Kagan 2003).

Although the I50V mutation is known to confer cross-resistance between amprenavir and lopinavir there are no data available on whether lopinavir-resistant virus is sensitive to amprenavir. A study is now underway comparing the ability of ritonavir-boosted amprenavir and saquinavir to salvage failing lopinavir therapy.

A study of over 1100 people who had taken PI therapy found mutations at E34Q, K43T, and K55R were associated with lopinavir resistance. Importantly, it was the presence of these specific mutations in the company of a cluster of other mutations which correlated with lopinavir resistance. Specific clusters associated with resistance to lopinavir were: E34Q with either L33F or F53L; K43T with I54A; K43T with V82A and I54V or V82A, V32I, and I47V; K55R with V82A, I54V, and M46I.

Other studies have reported:

• Five or more mutations at codons 10, 20, 24, 33, 36, 47, 48, 54, 82 and 84 predicted loss of response to lopinavir most accurately (De Luca 2003).

• The presence of mutations at codons 46L, 71V and 82A best predicted response to the drug, with mutations at codons 20M, 36I, 46L, 54 S/V, 71V, 73S and 82A contributing to resistance (Zolopa 2003).

• Mutations at codons K20MR, I54VL, G73SA, and I84V, or the presence of greater than nine protease mutations most strongly predicted resistance to lopinavir. The single mutation most strongly associated with resistance to L54V (Monno 2003).

• In HIV subtype C, I54V and V82A mutations have been associated failure of first-line lopinavir treatment (Conradie 2004).

• In vitro experiments have shown that lopinavir eventually selects the I84V and I50V/M46I mutants (Mo 2003)

Lopinavir response was reduced in patients with greater than 20-fold reduced susceptibility to lopinavir in the M98-957 phase III study, which evaluated the drug in PI-experienced patients. At week 24, 87% of those with four- to 20-fold reduced susceptibility had viral load below 400 copies, compared with 67% of those with 20- to 40-fold reduced susceptibility and 50% of those with greater than 40-fold reduced susceptibility (Kempf 2000).

Resistance to saquinavir

When boosted with ritonavir, lack of response to saquinavir is associated with two or more mutations at codons 24, 62, 82, 84 or 90 (Calvez 2003), according to a multivariate analysis of virologic response in 77 PI-experienced patients who received ritonavir-boosted saquinavir.

Resistance to indinavir

The genotypic pattern associated with loss of susceptibility to indinavir is not well defined (Szumiloski 2003).

Resistance to atazanavir

Test-tube studies show that a large number of mutations (at positions 10, 20, 24, 33, 36, 46, 48, 54, 63, 71, 73, 82, 84 and 90) are associated with resistance to atazanavir (Reyataz). The presence of five or more mutations will reduce the antiviral efficacy of atazanavir (Colonno 2003). However, the lopinavir-associated mutation L76V appears to be associated with only modest reductions in atazanavir susceptibility, despite the presence of high numbers of PI mutations, and its presence may provide an opportunity for the use of atazanavir in salvage therapy (Mueller 2004).

The development of atazanavir resistance in patients who have no prior PI experience is associated with the signature mutation I50L which does not confer cross-resistance to any other protease inhibitor. An analysis of 19 individuals who experienced treatment failure in studies of atazanavir found that all who took atazanavir as their sole PI developed the I50L mutation. Other mutations observed were at positions 71, 73 and 45. While these mutations significantly reduced susceptibility to atazanavir, there was very little impact on sensitivity to the other PIs (Colonno 2003b).

In contrast, people who took both atazanavir and saquinavir and developed resistance did not have the I50L mutation. In these patients, resistance to atazanavir required several substitutions including the I84V mutation (Colonno 2003b).

People who develop resistance to atazanavir on top of resistance to other PIs are likely to develop further loss of sensitivity to other protease inhibitors. Evidence to date suggests that the background pattern of mutations determines the evolutionary pathway of atazanavir resistance (Colonno 2002).

As with other boosted PIs, atazanavir/ritonavir has greater efficacy against resistant virus than atazanavir alone, due to higher drug concentrations (Coakley 2005). Resistance mutations that have been associated with the strongest response to atazanavir/ritonavir in PI-experienced people are 10F/I/V, 16E, 33I/F/V, 46I/L, 60E, 84V and 85V (Marcelin 2005).

When atazanavir resistance is assessed by loss of sensitivity rather than genotypic mutations, quite a high degree of cross-resistance may be present in patients who have experienced the failure of at least one PI. When samples with greater than 3.5-fold reduced susceptibility to approved PIs were tested for susceptibility to other PIs, atazanavir was amongst the most compromised agents(Schnell 2003).

An analysis of study 043 (atazanavir vs. Kaletra) found that response to atazanavir was related to the number of baseline protease mutations (three or more indicating reduced susceptibility), whereas response to Kaletra was not affected by the presence of five mutations. A less pronounced difference between the two drugs was found in an analysis of the 045 study, where ritonavir-boosted atazanavir was compared to Kaletra. Where no or one protease mutation was present, there was a trend towards greater viral load reduction in the atazanavir-treated group (although this result was not analysed for significance), but as the number of mutations increased, the degree of viral load reduction declined, with a trend towards poorer atazanavir response in the presence of five mutations (Zala 2003).

A study of 38 people enrolled in the Atazanavir Expanded Access Programme, two thirds of the whom were using ritonavir to boost atazanavir levels, found that patients with fewer than five resistance mutations had a better chance of virological success. Drug levels were also a key factor affecting treatment efficacy in this study (Gonzalez de Requena 2005).

Resistance to tipranavir

Tipranavir (Aptivus) has also been developed as a possible PI option for people with PI-resistant virus. Early laboratory and clinical studies were encouraging but more recent resistance data from a clinical trial suggests that there is some level of cross resistance between the established protease inhibitors and tipranavir (Gathe 2003; Yeni 2003). A poor response to tipranavir is associated with three UPAMs (mutations at codons 82, 84 and 90).

Resistance mutations associated with tipranavir are I15, E35D, N37D, D60E and A71T. Isolates with resistance mutations at codons 82T and 84 have also displayed reduced sensitivity to tipranavir. Other secondary mutations appear to be important in such cases for determining how much sensitivity is lost.

The RESIST study compared ritonavir-boosted tipranavir with other ritonavir-boosted PIs in over 1100 people who had previously taken PI therapy. Among people with any PI mutation, the response to tipranavir proved significantly better than responses to other PIs, no matter how many of the PI mutations a person had when the study started. A viral load fall of at least 1 log₁₀ after six months of treatment occurred in 41% of people taking tipranavir-ritonavir compared to 19% of people taking other boosted PIs. Tipranavir-ritonavir even had a better outcome than other PIs in people with the four protease mutations at positions 30, 82, 84, and 90, which were originally thought to impair response to tipranavir (Schapiro 2005; Valdez 2005).

There is some evidence that tipranavir resistance mutations at 82T and 82S predominate in subtype G, and that 82M may also be linked to tipranavir resistance (Camacho 2005). Another study reported a number of mutations occurring on non-B subtypes which contribute to resistance to tipranavir (Vandamme 2005).

Resistance to TMC114

Preliminary studies have not yet characterised the pattern of resistance mutations associated with resistance to TMC114, a new PI being developed by Tibotec. However, in vitro analysis of 1666 recombinant isolates showed that even in isolates with four mutations from the group D30N, M46 I/L, G48V, I50V/L, V82A/F/T/S, I84V or L90M, the observed reduction in TMC114 sensitivity was less than fourfold (De Meyer 2003).

A randomised study of ritonavir-boosted TMC114 in approximately 500 people who had previously taken treatments from the three main anti-HIV drug classes produced significant reductions in viral load. Of 40 people in the highest-dose group who had three or more primary protease mutations when they started the new PI, 19 had a viral load under 50 copies/ml 24 weeks later (Katlama 2005).

Natural variations

There is considerable natural variability in the protease enzyme. These variations are called polymorphisms, and may pre-dispose some people to protease resistance, although this has not been confirmed through research. Furthermore, these polymorphisms can make the interpretation of genotypic resistance testing very difficult.

These polymorphisms occur at high frequency in individuals infected with non-B subtypes (those prevalent outside Europe and North America). A study by the United States Centers for Disease Control found that 285 out of 300 protease sequences from drug-naive individuals contained at least one polymorphism seen in drug-resistant viruses (Pieniazek 2000). These polymorphisms have the potential to undermine responses to antiretroviral therapy (Turner 2003). Similarly, HIV-2 subtypes A and B frequently contain natural polymorphisms which may undermine PI therapy (Damond 2005; Pieniazek 2003). Some mutations in HIV-2 in response to PI treatment have not been seen in HIV-1 (Damond 2005).

Secondary protease mutations present before PI treatment have been shown to compromise response to treatment. An analysis of 248 drug-naﶥ patients who started treatment with a PI-containing regimen found that people who had a mutation at codon 36 at baseline were significantly more likely to experience treatment failure at even after allowing for the effect of NRTI mutations that might also be present. One weakness of this study is that no genotypic test was carried out to assess the contribution of new mutations that emerged on therapy, and whether a different pattern of primary resistance mutation emerged according to the presence or pattern of secondary mutations at baseline (Perno 2001).

Secondary mutations that are rare in treatment-naﶥ patients with HIV-1 sub-type B may represent the consensus sequence, or commonly found pattern, in HIV-1 subtype C, but there is little evidence that patients with subtype C have a compromised response to treatment. Phenotypic susceptibility was not significantly reduced in isolates with secondary mutations, and these viruses had reduced replication capacity compared with subtype B viruses without secondary mutations (Schapiro 2001).

Other reasons for drug failure

A number of studies have shown that early viral rebound is not necessarily the result of resistance to protease inhibitors. For instance, NRTI resistance can play a part in the failure of HAART (Monno 1999; Havlir 2000; Albrecht 2001; Descamps 2005). Evidence suggests that early rebound is more likely to be associated with resistance to the NRTIs (particularly 3TC) or an NNRTI than a PI, or other factors such as non-adherence or low drug levels in the blood. Detectable virus does not necessarily mean resistant virus is present; it may simply mean that the combination was not powerful enough to suppress HIV. PI resistance may only develop over time as multiple mutations associated with improved viral fitness emerge (Anton 1999; Atkinson 2000; Holder 1999).

The way a drug is metabolised in the body may reduce the effectiveness of a drug. There is considerable individual variation in metabolism, so some people may clear the drug from cells and the body more quickly than another person may. See Testing drug levels for further discussion of this issue.

One reason a person may have low drug levels is the molecule P-glycoprotein - a large molecule that actively pumps drugs and other molecules out of cells, including CD4 T-cells. The low bioavailability of the early version of saquinavir was due in part to P-glycoprotein pumping the drug from cells in the gastrointestinal tract. In contrast, ritonavir inhibits P-glycoprotein.

One study which investigated the effect of alpha 1-acid glycoprotein (AGP) on protease inhibitors reported that a four-fold increase in AGP reduced the anti-HIV activity of the PIs. Indinavir's effectiveness was only reduced by 2% but the activity of the other four PIs was reduced by between 30-42%. The efficacy of the PIs against wild-type HIV was not reduced (Zhang 2000). Researchers suggested that target drug levels be increased in individuals with PI resistance and high AGP levels. There is some evidence that P-glycoprotein does not contribute to NRTI drug failure (Speck 2002).

References

Anton ED et al. Development of HIV-1 genotypic resistance in patients failing efavirenz (EFV, DMP 266) plus nelfinavir (NFV) combination therapy (DMP 266-024). 37th Meeting of the Infectious Disease Society of America, Philadelphia, abstract 320, 1999.

Atkinson B et al. Correlation between human immunodeficiency virus genotypic resistance and virologic response in patients receiving nelfinavir monotherapy or nelfinavir with lamivudine and zidovudine. Journal of Infectious Diseases 182(2):420-427, 2000.

Bossi P et al. Polymorphism of the human immunodeficiency virus type 1 (HIV-1) protease gene and response of HIV-1-infected patients to a protease inhibitor. Journal of Clinical Microbiology 37(9):2910-2912, 1999.

Calvez V et al. Amprenavir genotypic resistance profile in protease inhibitor multi-experienced patients. Antivir Ther 4: abstract 42, 1999.

Calvez V et al. Identification of individual mutations in HIV protease-associated with virological response to lopinavir/ritonavir therapy. Antivir Ther 6: 64, 2001.

Calvez V et al. Clinically relevant interpretation of genotype for resistance to ritonavir (100 mg twice daily) plus saquinavir (800 mg twice daily) in HIV-1-infected protease inhibitor-experienced patients. Antivir Ther 8: S117, 2003.

Camacho R et al. Different substitutions under drug pressure at protease codon 82 in HIV-1 subtype G compared to subtype B infected individuals including a novel I82M resistance mutation. Antivir Ther 10: S151, 2005.

Cammack N et al. RO033-4649: a new HIV-1 protease inhibitor designed for both activity against resistant virus isolates and favorable pharmacokinetic properties. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 7, 2003.

Cane P et al. Resistance-associated mutations in the human immunodeficiency virus type 1 subtype c protease gene from treated and untreated patients in the United Kingdom. J Clin Microbiol 39: 2652-2654, 2001.

Chaillou S et al. Intracellular concentration of protease inhibitors in HIV-1-infected patients: correlation with MDR-1 gene expression and low dose of ritonavir. HIV Clin Trials 3: 493-501, 2002.

Churchill DR et al. The rabbit study: ritonavir and saquinavir in combination in saquinavir-experienced and previously untreated patients. AIDS Res Hum Retroviruses 15: 1181-1189, 1999.

Coakley E et al. Superior susceptibilities to both lopinavir and amprenavir in clinical isolates bearing the D30N mutation in protease after extended periods of viremia on nelfinavir-inclusive therapy. Antivir Ther 7: S112, 2002.

Coakley EP et al. Determination of phenotypic clinical cut-offs for atazanavir and atazanavir/ritonavir from AI424-043 and AI424-045. Antivir Ther 10: S8, 2005.

Colonno RJ et al. Identification of amino acid substitutions correlated with reduced atazanavir susceptibility in patients treated with atazanavir-containing regimens. Antivir Ther 7: S4, 2002a.

Colonno RJ et al. Amino acid substitutions that correlate with decreased susceptibility to atazanavir and other HIV-1 protease inhibitors. 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy San Diego, 2002b.

Colonno RJ et al. Activities of atazanavir (BMS-232632) against a large panel of human immunodeficiency virus type 1 clinical isolates resistant to one or more approved protease inhibitors. Antimicrobial Agents Chemother 47: 1324-1333, 2003.

Colonno R et al. Emergence of atazanavir resistance and maintenance of susceptibility to other PIs is associated with an I50L substitution in HIV protease. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 597, 2003b.

Conradie F et al. Failure of lopinavir-ritonavir (Kaletra)-containing regimen in an antiretroviral-naive patient. AIDS 18: 1084-1085, 2004.

Damond F et al. Polymorphism of the human immunodeficiency virus type 2 (HIV-2) protease gene and selection of drug resistance mutations in HIV-2-infected patients treated with protease inhibitors. J Clin Microbiol 43: 484-487, 2005.

D'Aquila RT et al. Drug resistance mutations in HIV-1. Top HIV Med 11: 92-96, 2003.

De Luca A et al. Improved prediction of virological response to lopinavir/ritonavir in salvage therapy using new interpretation rules of baseline genotypic resistance. Antivir Ther 8: S406, 2003.

De Meyer S et al. Antiviral activity of TMC114, a potent next-generation protease inhibitor, against >4000 recent recombinant clinical isolates exhibiting a wide range of protease inhibitor resistance profiles. Antivir Ther 8: S20, 2003.

Descamps D et al. Genotypic resistance analyses in nucleoside-pretreated patients failing an indinavir containing regimen: results from a randomized comparative trial: (Novavir ANRS 073). J Clin Virol 33: 99-103, 2005.

Dronda F et al. Cross-resistance to nelfinavir can be predicted by previous antiretroviral exposure in the absence of the D30N mutation. Seventh Conference on Retroviruses and Opportunistic Infections, San Francisco, abstract 729, 2000.

Friend J et al. Isolated lopinavir resistance after virological rebound of a ritonavir/lopinavir-based regimen. AIDS 18: 1965-1970, 2004.

Gathe J et al. Tipranavir/ritonavir demonstrates potent efficacy in multiple protease inhibitor experienced patients: BI 1182.52. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 179, 2003.

Gonzalez de Requena D et al. Atazanavir C_trough is associated with efficacy and safety: definition of therapeutic range. Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 645, 2005.

Grossman Z et al. D30N is not the preferred resistance pathway in subtype C patients treated with nelfinavir. Antivir Ther 7: S30, 2002.

Gulick RM et al. Indinavir, nevirapine, stavudine, and lamivudine for human immunodeficiency virus-infected, amprenavir-experienced subjects: AIDS Clinical Trials Group protocol 373. Journal of Infectious Diseases 183(5):715-721, 2001.

Gulnik S et al. Predicting cross-resistance profile of HIV protease inhibitors from biochemical analysis of active site mutants. Antiviral Therapy 4(supplement 1):45, 1999.

Hance AJ et al. Co-evolution and competition of viral populations with distinct resistance genotypes in patients failing treatment with protease inhibitors. Antiviral Therapy 7: S42, 2002.

Harrigan PR et al. Quantitation of lopinavir resistance and cross-resistance and phenotypic contribution of mutations shared with other protease inhibitors. Antivir Ther 6: S40, 2001.

Havlir DV et al. Drug susceptibility in HIV infection after viral rebound in patients receiving indinavir-containing regimens. JAMA 283: 229-234, 2000.

Holder DH et al. Virologic failure during combination therapy with expression of resistance-associated mutations in RT only. Sixth Conference on Retroviruses and Opportunistic Infections, Chicago, abstract 492, 1999.

Kagan RM et al. Emergence of a novel lopinavir resistance mutation at codon 47 correlates with ARV utilization. XII International Drug Resistance Workshop, Los Cabos, Mexico, abstract 49, 2003.

Katlama C et al. Efficacy of TMC114/r in 3-class experienced patients with limited treating options: 24-week planned interim analysis of 2 96-week multinational dose-finding trials. Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 164LB, 2005.

Kemper CA et al. Sequencing of protease inhibitor therapy: insights from an analysis of HIV phenotypic resistance in patients failing protease inhibitors. AIDS 15(5):609-615, 2001.

Kempf D et al. Identification of clinically relevant phenotypic and genotypic breakpoints for ABT-378/r in multiple PI-experienced, NNRT-naﶥ patients. Antiviral Therapy 5(3): 70, 2000.

Lastere S et al. Impact of amino-acid insertions in HIV-1 p6 PTAP region on the virological response to amprenavir in the NARVAL trial. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract P599, 2003.

MacManus S et al. GW433908 in ART-naﶥ subjects: absence of resistance at 48 weeks with boosted regimen and APV-like resistance profile with unboosted regimen. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 598, 2003.

Maguire MF et al. Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro. J Virol 76: 7398-7406, 2002.

Maguire MF et al. Emergence of resistance to protease inhibitor amprenavir in human immunodeficiency virus type 1-infected patients: selection of four alternative viral protease genotypes and influence of viral susceptibility to coadministered reverse transcriptase nucleoside inhibitors. Antimicrobial Agents Chemother 46: 731-738, 2002b.

Marcelin A-G et al. Clinical validation of atazanavir/ritonavir genotypic resistance score in PI-experienced patients. Antivir Ther 10: S9, 2005.

Martinez-Picado J et al. Phenotypic hypersusceptibility to multiple protease inhibitors and low replicative capacity in chronically HIV-1infected patients. Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 691, 2005.

Mo H et al. Characterization of resistant HIV variants generated by in vitro passage with lopinavir/ritonavir. Antiviral Res 59(3):173-80, 2003.

Monno L et al. Highly active antiretroviral therapy failure and protease and reverse transcriptase human immunodeficiency virus type 1 gene mutations. Journal of Infectious Diseases 180(2):568-571, 1999.

Monno L et al. HIV-1 phenotypic susceptibility to lopinavir (LPV) and genotypic analysis in LPV/r-naive subjects with prior protease inhibitor experience. JAIDS 33(4):439-447, 2003.

Mueller SM et al. Susceptibility to saquinavir and atazanavir in highly protease inhibitor-resistant HIV-1 is caused by lopinavir-induced drug resistance mutation L76V. Antiviral Therapy 9: S44, 2004.

Nijhuis M at al. Stochastic processes strongly influence HIV-1 evolution during suboptimal protease-inhibitor therapy. Proceedings of the National Academy of Sciences U S A 95(24):14441-14446, 1998.

Parkin N et al. Improving lopinavir genotype algorithm through phenotype correlations: novel mutation patterns and amprenavir cross-resistance. AIDS 17(7):955-961, 2003.

Paulsen D et al. Differentiation of genotypic resistance profiles for amprenavir and lopinavir, a valuable aid for choice of therapy in protease inhibitor-experienced HIV-1 infected subjects. Journal of Antimicrobial Chemotherapy 52: 319-323, 2003.

Perez-Elias MJ et al. Prevalence of universal protease associated mutations (UPAMs) in a large resistance database: impact on phenotype and virological response. Antiviral Therapy 8 (suppl1): S403, 2003.

Perno CF et al. Secondary mutations in the protease region of human immunodeficiency virus and virologic failure in drug-naﶥ patients treated with protease inhibitor-based therapy. Journal of Infectious Diseases 184: 983-991, 2001.

Petropoulos CJ et al. A mutation in human immunodeficiency virus type 1 protease, N88S, that causes in vitro hypersensitivity to amprenavir. Journal of Virology 74(9):4414-4419, 2000.

Pieniazek D et al. Protease sequences from HIV-1 group M subtypes A-H reveal distinct amino acid mutation patterns associated with protease resistance in protease inhibitor-naive individuals worldwide. HIV Variant Working Group. AIDS 14(11):1489-1495, 2000.

Pieniazek D et al. Protease sequences from HIV-2 subtypes A and B harbor multiple mutations associated with protease inhibitor resistance in HIV-1. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract P629, 2003.

Prado JG et al. Amprenavir-resistant HIV-1 exhibits lopinavir cross-resistance and reduced replication capacity. AIDS 16: 1009-1017, 2002.

Race E et al. Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients on combination therapies. AIDS 13: 2061-2068, 1999.

Resch W et al. Nelfinavir-resistant, amprenavir-hypersusceptible strains of human immunodeficiency virus type 1 carrying an N88S mutation in protease have reduced infectivity, reduced replication capacity, and reduced fitness and process the Gag polyprotein precursor aberrantly. Journal of Virology 76(17):8659-8666, 2002.

Rice H et al. Determination of clinically relevant phenotypic resistance breakpoints for indinavir/ritonavir-containing antiretroviral regimens. Antivir Ther 6: S61, 2001.

Ruffault A et al. Comparison of lopinavir/ritonavir resistance algorithms. Antivir Ther 7: S93, 2002.

Saah AJ et al. Treatment with indinavir, efavirenz, and adefovir after failure of nelfinavir therapy. J Infect Dis 187: 1157-1162, 2003.

Schapiro J et al. A preliminary report on the associations among clade C genotype, phenotypic drug susceptibility and replication capacity. Antivir Ther 6: S115, 2001.

Schapiro J et al. Effect of baseline genotype on response to tipranavir/ritonavir compared with standard-of-care comparator in treatment-experienced patients: the phase 3 RESIST-1 and 2 trials. Twelfth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 104, 2005.

Schmidt B et al. Low level of cross-resistance to amprenavir (141W94) in samples from patients pretreated with other protease inhibitors. Antimicrobial Agents Chemother 44: 3213-3216, 2000.

Speck RR et al. Differential effects of p-glycoprotein and multidrug resistance protein-1 on productive human immunodeficiency virus infection. J Infect Dis 186: 332-340, 2002.

Svicher V et al. Novel human immunodeficiency virus type 1 protease mutations potentially involved in resistance to protease inhibitors. Antimicrobial Agents Chemother. 49: 2015-2025, 2005.

Szumiloski J et al. Determination of an indinavir susceptibility cutoff for indinavir-ritonavir containing regimens. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 603, 2003.

Tamiya S et al. Amino acid insertions near gag cleavage sites restore the otherwise compromised replication of human immunodeficiency virus type 1 variants resistant to protease inhibitors. J Virol 78: 12030-12040, 2004.

Turner D et al. Novel drug resistance profiles in non-B subtype HIV-1 infections. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 144, 2003.

Vandamme A-M et al. HIV-1 subtype A1, C, F and G strains have a higher tipranavir mutation score than subtype B strains Antivir Ther 10: S152, 2005.

Valez H et al. Tipranavir/ritonavir 500mg/200mg BID drives week 24 viral load below 400 copies/mL when combined with a second active drug (T-20) in protease inhibitor experienced HIV+ patients. Third IAS Conference on HIV Pathogenesis and Treatment, Rio de Janeiro, abstract WeOa0205, 2005.

Yeni P et al. Correlation of viral load reduction and plasma levels in multiple protease inhibitor experienced patients taking tipranavir/ritonavir in a phase IIb trial. Tenth Conference on Retroviruses and Opportunistic Infections, Boston, abstract 528, 2003.

Zala C et al. Virologic determinants of 24-week efficacy of atazanavir with or without ritonavir in patients with prior failure on a protease inhibitor. Ninth European AIDS Conference, Warsaw, abstract F7/2, 2003.

Zhang XQ et al. The effect of increasing alpha 1-acid glycoprotein concentration on the antiviral efficacy of human immunodeficiency virus protease inhibitors. J Infect Dis 180: 1833-1837, 2000.

Zolopa A et al. Genotypic predictors of response to lopinavir/ritonavir in clinical practice. Antivir Ther 8: S415, 2003.