HIV Drug Resistance Workshop: entry and attachment inhibitors

New arsenals of compounds that focus on extracellular targets are currently under development – some further along the development pipeline than others. Even those in early, pre-clinical development are demonstrating their usefulness by elucidating membrane structure and mechanisms of co-receptor binding, viral entry and cellular fusion.

Compounds in development currently include the BMS attachment inhibitor (BMS-488043), the Pfizer CCR5 antagonist (UK-427,857), Anormed CXCR4 antagonist (AMD 3100) and very early in development a proposition by Merck for a synthetic antigen to elicit neutralising antibodies targeting gp41.

One key limitation of this class of compounds is that the efficacy of receptor binding is tropism-specific. In earlier HIV infection viruses are adapted to utilise the CCR5 receptor on CD4 cells. These viruses are known as NSI variants. In later disease, viruses adapted to utilise the CXCR4 receptor gradually become predominant. These viruses are described as SI variants.

The CCR5 and CXCR4 inhibitors that are most advanced in their development inhibit HIV binding to cells that express R5 or X4 respectively. However it is possible that the more promising of the attachment inhibitors will prove to be those that target the earlier stages prior to binding, in particular, the HR1 region of gp41; antibodies binding to this region do so only after the triggering of gp120 by CD4 activity that has involved prior co-receptor binding. Action at this site is not determined by tropism specificity and may therefore be able to target a broader range of viruses and thus, could be used in all patients, regardless of their stage of disease (NSI or SI) or viral tropism.

Amongst this target range, a promising candidate is the BMS small molecule attachment inhibitor which functions at the pre-CD4 form of gp120. Already in clinical development, this compound has been shown to work by binding to gp120 and interferes with gp120 and CD4 interaction. In vitro studies presented at this workshop highlighted potential sites of env resistance and inhibitor binding locations, including two common mutations at sites M434 and F423 whilst others were located at the CCR5 binding sites (C4 and V3). The latter sites may prove to be significant in that there is a key interaction between the V3 loop and the CD4 binding site C4; suggesting that this compound interferes with this critical part of the attachment process.

Another potential site for obstructing interaction and hence attachment, may be the V68A change near the N terminus of gp120 as well as changes further along the gp41 region. Despite the enthusiasm and scientific progress to date, a key caveat of this and other similar compounds will no doubt be the emergence of resistance, given that mutations in env are frequently selected. At the meeting, the presenters confirmed that rates of resistance emergence are likely to be similar to 3TC and NVP. Future studies will need to establish whether for example, the sites involved in interaction will be affected including substitution at W427V which has been profiled as a significant location for interaction. Furthermore, resistance pathways that may curtail the clinical utility of these compounds still need to be established (Lin).

The Merck HIV Antibody Discovery Team shared data from biochemical studies of a human monoclonal antibody designed to block HIV entry (D5 epitope). The study profiled the neutralising antibody effect on the HR1 region of gp41 – the site of action for T20. This study is interesting in that for the first-time a synthetic antigen is being used to generate ‘broadly’ neutralising antibodies. The study also provides proof-of-concept that HR1 of gp41 is accessible to human IgG and that an IgG directed at this region is able to block HIV entry. The researchers postulate the usefulness of this approach in the design of immunogens able to elicit antibodies in either the therapeutic or prophylactic vaccinations (Miller).

Studies of the Pfizer CCR5 antagonist (UK-427,857) currently in clinical development confirmed through intense resistance passaging techniques where increasing concentrations of the drug are added, that high-level resistance to this compound can be cultivated. In this study, three of the six viral cultures were able to acquire high-level resistance; two however, continued to utilise R5 co-receptors while the third virus showed reduced susceptibility and less interaction with R5 expressing cells. The authors conclude that resistance to UK-427,857 is in fact slow to emerge and in some variants did not emerge at all. Their studies confirm that for patients who have a predominantly R5 virus population, this compound may provide therapeutic benefit by conferring ‘considerable selective advantage’ at least in vitro (Westby).

Demonstrating potentially a broader target for attachment, the Canadian candidate AMD3100 is a co-receptor antagonist currently in Phase I/II development. In earlier studies, the researchers had found that several patients with dual tropic virus at baseline in fact went on to develop R5 tropic virus once on treatment. Interestingly since we know very little about evolution of co-receptor expression and subsequent switching from NSI to SI viruses, this study was intended to determine whether dual-tropic viruses were mixed mono-tropic (virus populations that contain both X4 and R5-tropic viruses) or dual-tropic (viruses expressing both X4 and R5 receptors).

To do this, the researchers cloned gp160 sequences at different time points. The virus population was studied at day 0 and day 11 of monotherapy and days 18 and 39 off-treatment characterised according to co-receptor tropism of between 25-40 env clones. Of the three patients who exhibited dual-tropic virus before treatment and switched to R5 virus on treatment, one patient demonstrated co-receptor utilisation of R5 at 66% and X4 at 34% at baseline, but at Day 11 of treatment, 100% of this viral population transferred to R5 and remained so at Day 18 off-treatment. However, the virus had mutated back to the original baseline dual-tropic profile at Day 39 off-treatment, suggesting that the switch to R5 is not maintained once treatment is withdrawn.

Similar patterns were noted for the second patient, although with some variations in the tropism profile sustained at Day 39 off-treatment. Interestingly, the final patient not only remained predominantly R5 throughout the study, but completed Day 39 with 100% R5 virus after having been 67% R5 tropic at baseline (Day 0). Of the env mutations observed under selective drug pressure, the profile of sequences matched pre-existing variants already established at baseline. This study confirms that X4 and dual-tropic viruses can be suppressed and the relative proportion of R5 increased, in one patient to 100% - higher than observed at baseline. This observation may prove to be significant in enabling patients to retain the R5 (NSI) virus, thereby suppressing X4-tropic variants and so potentially deferring disease progression associated with the presence of X4 viruses (Huang).

Data relating to the mechanism of action and resistance of T20 (Fuzeon) continues to emerge from the combined laboratories of Trimeris and Roche. Several studies at the workshop highlighted the impact of T20 resistance and progress in diagnostic techniques including envelope entry assays. One such assay in development by a national Canadian coalition including McGill and University of Toronto involves the assessment of the entire sequence of gp41, whereas existing technologies evaluate mutations in only 10 gp41 amino acid positions. The objectives of the study were to determine whether resistance evolution could be detected at locations of gp41 not previously characterised, if pre-therapy polymorphisms could be identified and whether either of these variables could be linked to T20 treatment failures.

The patient cohort included 400 T20 naïve patients and 44 patients assessed before and following treatment failure. Polymorphisms were identified in both B and non-clade B patients. In Clade B, insertions most commonly located were at positions 3 and 215, whilst in non-B clades, natural polymorphisms were detected throughout the gp41 sequence – 10 polymorphic changes in clade B patients and 39 changes in patients with clade A/E virus. A number of mutations already defined by expert panels (IAS-USA) were confirmed and two new mutations identified at 40H and 45M, although it should be noted that these are located within the already defined 36-45 region.

The cohort also confirmed that following cessation of treatment with T20, reversion to WT virus occurred within 30 days. This study is useful in that it adds to the diagnostic resource that will be needed as T20 establishes itself in the clinic. However, we do not yet know whether polymorphisms in gp41 will prove to be clinically significant either for patients with clade B, non-B or for both. The mapping of new mutations remains important for patients on T20 who by default have fewer options for switching therapy. However, these will need to be validated and subsequently incorporated into expert interpretation panels(Walmsley)

The findings from this study of reversion to susceptible virus were further corroborated by a combined Trimeris/Roche study assessing viral kinetics following withdrawal of chronic T20 treatment. Patients were given T1249 as replacement therapy for ten days with the option of continuing or discontinuing T20 and remaining on their failing background regimen. Genotypic and phenotypic analysis was performed whilst patients were on a failing regimen that contained T20 but prior to starting T1249 and then again after restarting or discontinuing T20 therapy. All 18 patients in the study had significant experience of T20 with a median of 61 weeks on a failing T20-containing regimen. Of these, ten patients interrupted therapy and eight remained on T20 treatment (median of 114 days and 82 days respectively).

As may be expected patients who remained on fusion inhibitor therapy did not experience an increase in viral load, whereas patients who interrupted had a median increase of HIV-1 RNA of +0.21 log10. Decreases in T20 IC50 >10 fold were seen in none of the eight patients who resumed T20 but observed in six of the ten patients who did discontinue therapy (interrupted for a median of 114 days: 46-230 days). The length of interruption correlated with changes in T20 susceptibility with the shortest discontinuation period (46-57 days) resulting in no changes in T20 susceptibility. Genotypic changes were associated with reversion to T20 susceptibility in patients who stopped and then started therapy. The study demonstrates that decreased viral fitness most likely explains the observation of interrupted therapy leading to the emergence of more susceptible virus. Tentative conclusions from this and other corroborative studies would suggest that for patients with T20 resistance, there may be a benefit to continuing on therapy on the basis of a impaired viral fitness, but that the option of interrupting T20 to allow genotypic reversion may also be a viable clinical option (Miralles).

An interesting study from Cleveland considered the combined impact of changes not only in protease (PR) and reverse transcriptase (RT) but the overall impact of mutations in pol including env sequence. This has direct significance for patients on T20 who are likely to have experience of 3-class regimens when starting T20. Five multi-drug resistant (MDR) strains of HIV from four different subtypes (A, B, D and CRF01_AE) were selected. Full-length gp160 recombinant viruses were generated and specific gp41 mutations added to the reference sample. They were able to characterise a number of substitutions at both previously identified locations (V38E and N43D, V38K) and novel sites (Q40K, L45Q). Whilst viruses with these mutations showed reductions in susceptibility to T20, no significant reductions in RC were observed. Viruses with only the V38K and Q40K (novel mutations) did however show a marked impairment of RC. The study confirms that T20 resistance is influenced by baseline genetic background and as shown by others, pre-existing compensatory mutations in env have only a minimal impact on viral fitness (Quinones-Mateu).

What were the key messages from this year's Resistance Workshop?

This year's workshop heard more about the clinical relevance of replication capacity and hypersusceptibility, and began to debate the potential therapeutic exploitation of interactions between resistance mutations.

What sort of resistance is occurring in clinic populations, and what does this tell us about clinical practice today?

The threat of nevirapine resistance is forcing a rethink of strategies to prevent mother to child transmission. The workshop learnt more about resistance patterns, and why differences in HIV-1 subtype may need to be taken into account when thinking about preventing mother to child transmission.

As treatment access expands and the proportion of patients in Europe with non-B subtypes grows, understanding differences in resistance patterns between B and non-B HIV subtypes becomes more and more important.