Understanding developments in HIV drug resistance for new drugs and targets — atazanavir, fusion (T-20) and receptor inhibitors

Mike Youle, for NATAP

Unless otherwise stated, all references are to the Programme and abstracts for the 2nd European HIV Drug Resistance Workshop: from basic science to clinical implications.

Understanding developments in HIV drug resistance for new drugs and targets — atazanavir, fusion (T-20) and receptor inhibitors

The annual European gathering of resistance aficionados took place recently on a hill above the Vatican and conformed to the best of Italian characteristics: good food, exquisite style and a complete disregard for time-keeping. I chaired a session that started 90 minutes late but since the presentations and discussion were so engrossing no one but the most anally retentive cared a jot.

The attachment of HIV to the cell is a potentially very attractive proposition since the likelihood of toxicity from these compounds is low and an interesting subject of discussion at this conference. The meeting opened with an excellent overview of “Research on Anti-HIV Resistance in Europe” by Anne-Mieke Vandamme who has just finished editing the new European Guidelines for Resistance testing which will be available soon. She spoke of the central problem that HIV therapy effectively treats the condition but a consequence is a rise in resistance since even the best agents leave a window of replication and inadequate treatment or poor adherence lead to a selection of resistant strains which can then be transmitted, more of which later. So what are the answers? Certainly one is to develop new agents and she showed various lists of the new drugs in development, which appear to be licensed at the rate of 1-2 per year. But from her perspective as a virologist the newer therapies have inherent problems since most are aimed at existing targets and thus have potentially reduced efficacy through cross resistance.

In the next five years it is possible that we will have seven new NRTI’s, five NNRTI’s, five PI’s, nine entry inhibitors and perhaps three-four other agents but in Anne-Mieke Vandamme’s view drugs are not coming fast enough to keep up with the rising tide of resistance especially in highly treated populations where 3 class resistance is already approaching 40-50%.

She showed the major groups who are working on HIV drug resistance but bemoaned the fact that much of the peer-reviewed money has been for establishing networks but not for actually conducting research projects. Apart from the ANRS in France, other European countries appear to feel that the pharmaceutical industry should provide the major part of funding for such research, which of course brings its own difficulties.

An excellent aspect of Anne-Mieke Vandamme’s talk was a case study in which clearly a mixture of an inexperienced or ill informed physician had prescribed a poor regimen that had been wrongly taken by the patient, leading to pan-resistance when the treatment had been started well after the 1996 HAART threshold. This resulted in a salvage treatment scenario for no good reason, emphasising that most therapy failure, and ipso facto problematic HIV care, is based on bad medical decisions. Leaving antiretroviral treatment to experts in centres of excellence would probably reduce this. She finished her presentation with a few suggestions of areas of importance such as the development of fusion inhibitor (T20) and other entry inhibitor assays, further study of the epidemiology of drug resistance and establishing the role of resistance testing of proviral DNA.

The first subject discussed in the main body of the meeting was the Epidemiology of Primary Drug Resistance and Viral subtypes. Sally Blower from USFC gave an overview of modelling work she has done of drug resistance transmission in Africa, the potential for which has been a traditional argument against the widespread use of antiretrovirals in resource poor settings. She estimates that of the 34-46 million infected in the subcontinent, 6 million need immediate treatment, which the UNAIDS 3 x 5 programmes is attempting to achieve. The models that were published by her last year in Science estimate that by 2006 40% of treated individuals will have drug resistance to some degree. She then moved focus to consider the potential for HIV therapy to eradicate the epidemic by preventing transmission, a kind of secondary chemoprophylaxis. Her conclusions were that unlike the developed countries where a high proportion of those infected are on antiretrovirals, it is unlikely that the treatment levels seen in Africa will have any chance of slowing the epidemic.

Jan Albert of the Swedish Institute for Infectious Disease Control broached the difficult problem of transmission of drug resistance [1]. An expert panel is to be convened to try to address some of the difficulties in defining the field, since nothing currently exists to help the virologist identify whether or not drug resistance transmission has occurred, for there are no guidelines of interpretations systems available. He raised the difficulty of treating the presence of singleton mutations (single codon changes such as K101E or M36I) as evidence of transmission since few treated patients carry single mutations, and he posed the question “Who are the source patients for these infections?” He cited two studies where half of the purported drug transmissions were singleton mutations and suggested that either these were natural variants or a result of the reversion of more complex resistance patterns if indeed they were transmissions.

Further epidemiologic data were presented by David van der Vijver on behalf of the CATCH study team, which tracked the occurrence of resistance in 2208 subjects across 19 European countries [2]. While the prevalence of resistance was higher in B subtypes (13%) versus those harbouring non-sub-type B viruses (5%) this sub-analysis which merely looked at those from the main cohort who had been infected for less than a year revealed an increase in the proportion of individuals with non-B viruses which rose from 17% in the 1996-9 period to 28% between 2000 and 2002 (P<0.001).

The breakdown showed that 95% of MSM had B, whereas only 72% of IDU patients and 58% of heterosexuals carried this sub-type. The effect of immigration, especially more recently, and the rates of non-B virus were discussed as well as the variable pen trance of treatment into various transmission groups, which may explain these findings.

Deenan Pillay from University College London gave a poster review of this section and clearly laid out some of the problems associated with the current ways of looking at the subtype issue. In 12 abstracts, looking at subtypes, he found six methodologies varying from comparison to a standard strain through, amino-acid weighting within sequences of the virus to formal phylogenetic testing. In addition, the proportion of non-B using these different tests varied widely from 45% in Greece down to 8% in Germany.

Next was a section on new agents and their pathways to resistance. Rich Colonno from Bristol Myers Squibb (BMS) gave an elegant presentation on the available data for the recently licensed protease inhibitor atazanavir [3]. He first listed the primary and secondary mutations known to be associated with reduced sensitivity of the virus to atazanavir. These included the usual suspects (primary G48, V82, I84 and L90, along with so far unique I50L). Resistance was defined as the presence of the I50L or a greater than 2-fold phenotypic change from baseline to >2.3-fold. In the naïve studies all individuals seem to preferentially follow the I50L pathway to resistance which seems to induce hypersensitivity of around 10% to other protease inhibitors, which of course may argue for the use of atazanavir up front to allow some form of sequencing, although previous history has not been kind to this strategy and the argument for Kaletra has always been that no primary resistance occurs, so why not use it first line.

He next showed the results of the experienced studies and clearly the virus takes another pathway more frequently here with only 36% (13) individuals in the 043 protease inhibitor experienced study developing I50L, while the rest presented with standard PI mutant virus, which of course showed cross resistance to other PIs. The same story has emerged for the ritonavir boosted second line PI studies such as 045 where 21% (5) had I50L compared to 79% (19) with other mutation patterns. Interestingly, none of the subjects who received atazanavir combined with saquinavir and who showed a sub-optimal response compared to the ritonavir boosted arms followed the I50L pathway and all revealed other more commonly seen PI mutation patterns. Finally, hyper susceptibility to other PI’s was only found in the setting of I50L. In the questions that followed the talk, Françoise Brun-Vèzinet remained unconvinced that enough data were yet available on boosted atazanavir to be clear on the route the virus would take to resistance under atazanavir, and Yasmin Halima asked the very pertinent question as to the effect of drug levels on this pathway. It has been received wisdom from BMS that therapeutic drug monitoring is not necessary with atazanavir but I feel that many of us who use this tool within clinical practice for other PI’s would not necessarily agree with this, and in fact a study linking drug levels with outcomes was presented by Soriano and his group at CROI two months ago suggesting that there is a link.

Continuing the theme of assessing new compounds, there was a fascinating talk by Rafael Nájera on natural resistance mutations to fusion inhibitors (T-20) and polymorphisms in the gp41 sequence of both B and non-B subtypes from patients in Spain [4]. The group studied the de novo resistance to several fusion inhibitors (T-20, 5-helix, C-34 and RPR 103611) in 170 individuals of whom 19 were primary infections and 131 were on HAART. RNA was extracted from plasma samples and sequencing undertaken of gp41 by nested PCR, then resistance mutations were assessed within the heptad repeat 1 and 2 regions which are blocked by fusion inhibitors. Polymorphisms were found in 42.3% of subjects (28.5% in B subtypes; 72.2% in non-B sub-types) and mutations which have been associated with resistance to T-20 in 11.2% (3.5% on HAART/T20; 7.6% on T-20 functional monotherapy). The N42S polymorphism had the characteristic of increasing susceptibility to T20 in B sub-types.

This study raises some concerns that T-20 naïve individuals may have a variable response due to innate conformational changes in gp41 due to these mutations and that resistance testing may be helpful in predicting response to the drug. Further work in this area was shown by a group from the retrovirology group in Centre de Recherche Public-Santé in Luxembourg [5]. They created a recombinant virus into which was inserted a mutation I37V which has been associated with resistance to T-20 both in the test-tube and after giving the drug as monotherapy. The findings were that the virus was less fit but did not exhibit a reduced susceptibility to T-20 and thus of itself did not result in T-20 resistance.

A final piece of the story was provided by Poveda and co-workers from Madrid, who examined the consequences of T-20 therapy in multi-drug resistant patients who received T-20 as part of a salvage regimen. Of eight patients, all of whom experienced a decrease in viral load >0.5log after starting their new regimen, one remained suppressed <50copies/mL while the other seven rebounded rapidly after commencing treatment. Six of these exhibited a N43D mutation and showed between 15 and 143-fold resistance to T-20, two subjects also had a G36V or D leading to high level resistance. These patients were also noted to have a wide range of baseline susceptibilities to T-20 (IC50 range from 20-400) and this may be important in the future assessment of suitability of subjects for T-20 or other fusion inhibitor drugs. This is definitely a field in which we are working with little data at the moment and as this increases a clearer picture will emerge of how to evaluate and interpret resistance to this new class of drugs.

Further discussion of entry inhibitor resistance was provided by Francois Clavel, who first talked about receptor blockers and the potential pathways to resistance, specifically what happens to X4 virus when R5 is inhibited and how much X4 is required to outgrow R5 suppression. A difficulty remains that the current assays are at best semi-quantitative with a rather uncertain level of sensitivity, which precludes accurate prediction of what actually happens. Some data exist that suggest that in the presence of a receptor blocker you can overcome the effect by adding more virus and that artificially producing V3 loop mutations allows resistant viruses to use low density receptors preferentially. Some of the receptor blockers (SCHC for instance) may not bind but rather alter the binding characteristics of the virus.

With regard to the fusion inhibitors, of which really enfuvirtide (T-20) is the only one for which we have long-term clinical data, he described the very variable levels of resistance to fusion inhibitors between isolates and the reported effect of mutations on HR2, notably S138A (and from another study 113, 126 and 135) having an effect on HRI where T20 binds. These codons are actually opposite the area of gp41 (codons 36-43) which are at the site of action of T20 so in structural terms this makes sense. He also presented a table of report variation in natural susceptibility of HIV strains to a raft of entry inhibitors. These range from 1-23 fold for PRO-542 to 1-10,000 fold for the BMS-806 compound that has now been superseded by a new construct. Finally, isolates show at least a 2-log variation in susceptibility to T-20 although this did not appear to be clinically significant from the TORO studies.

He then tried to explain these phenomena in terms of the mode of action of the current agents and concluded that the receptor or target density, as well as the speed of the action in the window of opportunity during the events leading to fusion, predict the effect of the drugs. He concluded “HIV entry is not just another target since all envelopes are escape mutants” and the high variability of this region will cause difficulties. In addition, he commented that the current assays we have specifically for R5 and X4 are in their infancy. We obviously have a lot to learn in this new area of HIV control.



  1. Albert J, Guidelines for identification of transmission of drug resistant HIV. Abstract 1:P1.1
  2. Van der Vijver DAMC, Wensing AMJ, Op de Coul E et al. Increasing prevalence of HIV-1 non-B subtypes across Europe from 1996-1999 to 2000-2002; results from the CATCH study. Abstract 3:P1.3
  3. Colonno R, McLaren C and Kelleher T. Pathways to atazanavir resistance in treatment-experienced patients on atazanavir containing regimens. Abstract 28:P3.1
  4. Carmona R, Munoz M, Perez-Alvarez L et al. Natural resistance mutations to fusion inhibitors and polymorphisms in gp41 sequence of recombinant forms, non-B and B subtypes from HIV-1 infected patients in Spain. Abstract 29:P3.2
  5. Roman F, Ammerlaan W, Plesseria JM et al. The gp41 mutation I37V does not lead to enfuvirtide (T-20) resistance. Abstract 30:P3.3

Links to other websites are current at date of posting but not maintained.