Integrase inhibitor resistance and cross-resistance: weighing viral fitness and the option to benefit from second-generation compounds
Simon Collins, HIV i-Base
Several research groups presented results on generally small groups of highly treatment experienced patients who are no longer responding to raltegravir-based regimens.
The key questions for these patients who already have limited options involves identifying when the risks of remaining on a less than suppressive treatment outweigh any residual benefits from treatment.
- Do integrase inhibitors (INIs) have class properties that are stronger than individual drug profiles (generally like NNRTIs or PIs)?
- Does resistance accumulate slowly enough for reduced viral fitness to have a clinical benefit?
- Does the risk of accumulated mutations compromise the future option to benefit from second generation INIs?
An understanding of the integrase resistance, first reported at last years meeting, emphasised that primary resistance follows two main pathways – based on either Q148H/R/K or N155H, leading to >10-fold resistance, with one group this years reporting that these mutations are not found on the same genome. Indeed, four distinct pathways appear to develop, with different implications for viral fitness. This year Y143R/C emerged in several studies as a further primary mutation.
While resistance generally has an impact on reducing viral fitness, and immunological benefits were reported as continuing 3 and 6 months after treatment failure, questions were asked to each presenter about the degree to which patients maintained on a failing integrase-based regimen may be compromising their options to use second-generation integrase compounds.
This issue will not be resolved until they are greater data available on the viral responses of patients who stop integrase treatment – and this is currently limited to single case reports.
Michael Miller presented Merck’s longitudinal genotyping and cloning analysis from 35/133 patients (28%) failing raltegravir at 48 weeks in their Phase II study. 
Three additional patients had showed no INI changes. Of the 35, 2 subsequently resuppressed their viral load, leaving 33 patients with a second genotype. Of these, a further 2 resuppressed and 4 were lost to follow-up, 4 remained stable with Q148H and G140S and 23 provided data from a third genotype.
At first failure only 9 patients had single mutations, with most having 2 or more, and these increased in number over time. The genotype at first failure showed 20 patients with mutations at Q148 + combinations and 14 with N155 plus combinations. No patients failed with mutations at both these key sites. One patient failed with Y143R alone.
Over time, four pathways were reported: i) a consistent preference for the Q148H at each stage, including Q148H/G140S (n=13) that remain stable; ii) patients with N155H alone, which generally developed to Q148 (n=5); iii) mixed N155H/Q148H viral populations that resolved to Q148H over time, often with G140S (n=7); and iv) patients with other mixed patterns including N155H but who accumulated additional mutations but not Q148 (n=7).
Q148 viruses, especially with secondary mutations, displayed the greater levels of resistance and the least benefit from impaired fitness.
Addressing this small dataset, without details on the time of sampling, Miller was optimistic that second generation Merck compounds would be active against Q148 and N155, but also did not expect to see pharmacologically acceptable formulations available for at least several years.
Most importantly, he also recognised that there were no data to support maintaining raltegravir in patients who were virologically failing treatment.
Signe Fransen and colleagues from Monogram, also working with the Merck team presented results from a subset of 69 samples from the Phase III Benchmrk studies. 
Nine patients had mutational changes at both Q148 and N155, but the changes at each point were mixtures and they were never found on the same genome. Secondary mutations – E92Q with N155H, and G140S(A) with Q148R(H/K) – were related to the primary resistance pathway. A few patients were reported with E92Q but without changes at N155 or Q148.
Four patterns in this analysis broadly followed the Miller study: i) Q148R(H/K) with or without additional changes including N155H and Y143R/C; ii) N155H with or without additional mutations, including Q148R(H/K) and Y143R/C; iii) Y143R/C with or without changes at N155 or Q148; and iv) 1 patient with no primary mutations.
Clonal analysis from these patients showed a variable impact of mutation patterns on viral fitness. Both Q148 and N155 reduced replicative capacity and this was either further reduced or, in some cases improved, depending on the secondary mutation pattern.
However, there is unlikely to be any significant clinical relevance from what are likely to be transitory changes in fitness, especially if a trend to developing Q148 is confirmed, as this mutation has little impact on fitness.
Clinical experience from raltegravir use was also provided by several independent research groups.
Steven Deeks group from University of San Fransisco, presented results from 13 patients who remained unsuppressed on raltegravir-containing regimens. 
These were multiple experienced and advanced patients (baseline CD4 count was 66 cells/mm3) followed for a median of over 12 months. Most notably, CD4 increases persisted despite virological non-suppression (with increases of +37 and +71 cells/mm3, at 3 and 6 months respectively).
Three patients with intermittent adherence in this group lacked mutations, but otherwise G140S+Q148H and T97A+Y143R patterns were seen, and they were associated with high-level phenotypic resistance.
One patient, who only showed isolated N155H, interrupted only the raltegravir componant of treatment while maintaining their background regimen. Plasma levels initially remained stable, suggesting an effect of viral fitness, but they subsequently rebounded by over 1 log as the proportion of raltegravir-associated mutations declined in the absence of selective drug pressure.
Christine Katlama from Pitie-Salpetriere Hospital, Paris, reported on a cohort of 50 treatment-experienced patients who started raltegravir between September 2006 and November 2007. 34 patients successfully suppressed viral load to <40 copies/mL by week 24. 11 patients had low-level viraemia >40 – <400 copies/mL and 3 had persistent viraemia >400 copies/mL.
4/13 patients with genotype results showed integrase mutations: G140S+Q148H (n=2), N155H (n=1) and a switch fromN155H to G140S+Q148H (n=1).
Several studies also addressed cross-resistance between raltegravir and elvitegravir, and no researchers either directly involved in these studies or in the general discussions expected patients who fail on one of these drugs to benefit from subsequent use of the other.
Olivia Goethals and colleagues from Tibotec used selected in vitro emergence of resistance to raltegravir and elvitegravir and site directed mutagenesis of selected mutations against a panel of integrase inhibitors. 
Q148R was selected by both raltegravir and elvitegravir and conferred resistance to other compounds. The researchers concluded that Q148H, together with E92Q and T66I, which were both also selected by elvitegravir, should be considered to confer class-wide resistance.
The discussions on cross-resistance were closely tied to interest in second-generation integrase inhibitors, and awareness of the pipeline is important for patients who have already devloped integrase resistance. Merck say they have already designed molecules with activity against 148 and 155 mutations – and that rational drug design should also be able to overcome changes at position 143. The bottleneck is overcoming pharmacokinetic problems of delivery and tolerability though, and Daria Hazuda also estimated that they are still likely to be at least 2-3 years away from clinical studies.
Finally, a study from Anne-Genvieve Marcelin and colleagues from Pitie-Salpetriere Hospital, Paris, compared integrase sequences from 72 clade-B and 66 CRF02-AG strains for the difference in naturally occurring polymorphisms. 
While several studies have reported that polymorphisms in subtype B infection are not limiting responses in integrase-naive patients, including two studies at this workshop [7, 8], there are little data from non-B subtypes.
They found 13 amino acid changes that could affect the functional properties of integrase with clustering suggesting these may include compensatory mutations, concluding that virological response to integrase inhibitors by viral sub-type should be studied in clinical trials.
This focus on resistance should be seen against the background of raltegravir also producing some of the strongest treatment responses in multiple-experienced and advanced patients.
For patients failing integrase-based treatments though, these questions are critical. Unlike the US, which needs to wait for a validated integrase resistance test, integrase resistance is already being sequenced in many UK and European labs, and is being validated as part of a European quality control programme.
Documenting resistance history for selection of future drugs is important. The limited data, and expert opinion from the researchers involved with these compounds suggest no specific benefit from remaining on integrase inhibitors once viral load has rebounded, and that the risk of accumulating mutations will compromise use of future INIs and that this risk should be taken seriously.
The data on viral fitness suggests any benefit is likely to be short term, and unlikely to outweigh the risk from accumulating further resistance, especially once Q148 changes have occurred.
- Miller M et al. Longitudinal analysis of resistance to the HIV-1 integrase inhibitor raltegravir: results from P005, a Phase 2 Study in treatment-experienced patients. XVII IHDRW 2008, Sitges. Abstract 6.
- Fransen S et al. Loss of raltegravir susceptibility in treated patients is conferred by multiple non-overlapping genetic pathways. XVII IHDRW 2008, Sitges. Abstract 7.
- Hatano H et al. Virological and immunological outcomes in a cohort of patients failing integrase inhibitors. XVII IHDRW 2008, Sitges. Abstract 10.
- Katlama C et al. Virological evolution on HIV treatment-experienced patients with raltegravir-based salvage regimens. XVII IHDRW 2008, Sitges. Abstract 11.
- Goethals O et al. Resistance mutations in HIV-1 integrase selected with raltegravir or elvitegravir confer reduced susceptibility to a diverse panel of integrase inhibitors. XVII IHDRW 2008, Sitges. Abstract 9.
- Marcelin A-G et al. Natural variation of integrase sequences form subtype B and CRF02-AG HIV-1 antiretroviral naive patients and possible effect on susceptibility to integrase inhibitors. XVII IHDRW 2008, Sitges. Abstract 63.
- Buzon J et al. Fitness progression and phenotypic susceptibility to raltegravir of HIV-1 integrase are not restricted in long-term HAART treated patients. XVII IHDRW 2008, Sitges. Abstract 76.
- Ji H et al. Tracking HIV-1 integrase polymorphisms from the pre-antiretroviral therapy era up to the introduction of integrase inhibitors. XVII IHDRW 2008, Sitges. Abstract 78.