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Report from International Workshop on HIV & Hepatitis Virus Drug Resistance 5-9 June 2012, Sitges, Spain
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Written for NATAP by Mark A. Wainberg, PhD, McGill University AIDS Centre, Jewish General Hospital, Montreal, Canada
International Workshop on HIV & Hepatitis Virus Drug Resistance
5-9 June 2012, Sitges, Spain
The Workshop on HIV and Hepatitis Virus Drug Resistance and Curative Strategies was held in Sitges, Spain between June 5-8, 2012. The conference was a great success and was attended by several hundred people. Much more of a focus was placed this year on Hepatitis C Virus and Hepatitis B virus than in previous years in relation to the growing bodies of knowledge relative to these agents that have been obtained in recent years. The conference dealt with a wide variety of subjects in the fields of each of HIV, HBV, and HCV, including curative strategies.
Persistence, Reservoirs, and Elimination Strategies
This topic was one of the major highlights of the conference in view of the fact that it dealt with the potential to actually effect a cure of HIV in years to come. There is now a critical mass of research activity in this field with many investigators realizing that viral eradication is probably the only truly sustainable means of dealing with the HIV epidemic on a worldwide basis. This was adequately pointed out in a plenary lecture by Dr. Sharon Lewin of Melbourne, Australia who stated that the number of HIV cases diagnosed each year continues to grow and that, on a worldwide basis, more than twice as many people become HIV infected each year as are able to gain access to therapy. This creates a direct need for eradication, in spite of the fact that many individuals, if not most, who live in resource developed setting do have access to antiretroviral therapy. Dr. Lewin pointed out that the problem of HIV persistence is such that the virus can apparently survive in multiple tissues at levels below limits of common detection as determined by viral sequencing of plasma by ultrasensitive quantitative methods. In fact, a great deal of research now indicates that the virus can survive at far lower levels than previously thought, as determined by single copy assays for detection of viral RNA and DNA and by other sensitive methodologies. In particular, central memory key cells seem to be essential to the ability of HIV to survive in long-lived reservoirs. A number of investigative groups are now attempting to target such reservoirs through the use of histone deacetylase(Hdac) inhibitors as well as through other approaches. Work being carried out also involves use of interleukins such as IL7 to try to stimulate latently infected cells and to try to confront the problem of homeostatic proliferation of latently infected cells as well as viral proliferation. Dr. Lewin pointed out that PD-1 has been identified by some labs as a key negative regulator of HIV activation that plays an important role in the maintenance of latency. Accordingly, strategies that target PD1 could represent small molecular approaches aimed at blocking HIV sequestration are being studied.
Dr. Lewin pointed out that there are many arguments against the notion of ongoing residual replication in reservoir cells. One strong argument is that patients who seem to be fully suppressed while on antiretroviral therapy do not seem to demonstrate evolution of viral RNA or DNA within their tissues. As an example, there does not seem to be any change in the profile of drug resistance mutations during the time that someone has been virologically suppressed, as might be expected if active viral replication were to have occurred during this time. However, there are also conflicting reports on this topic with some investigator reporting that low level viral replication does in fact occur on a routine basis. As an example, many patients who seem to be fully suppressed may occasionally have blips in regard to the presence of higher levels of viral RNA in their circulation. Although the full implications of this are still unknown, there is a good correlation between viral persistence and immune activation in patients remaining on anti-retroviral therapy. Of course, in this context, full tissue penetration of drugs is a major issue, as is the need to seek out individuals who may demonstrate evidence of very low level viral replication after having received antiretroviral therapy, even though this seems to be a fairly rare event. It is possible that there may be some association in people who are low-level producers of virus throughout their lifetime in regard to certain HLA haplotypes such as B27 and B57. However, people who manage to control plasma viremia after antiretroviral therapy has been withheld, although exceedingly rare, do not seem to possess any consistent HLA haplotypes.
Dr. Lewin focused on the use of Hdac inhibitors that could turn on genes and that might result in elaboration of newly made virus. The goal of such approaches would be to ensure that patients remain on antiretroviral therapy during time of exposure to Hdac inhibitors, such that all residual virus might be eliminated from the body. A number of clinical trials on this topic are now in progress. Certain of these involve compounds already approved for a certain type of cancer such as cutaneous T Cell leukemia and one such compound is Vorinostat (SAHA). One potential problem is that numbers of these compound have tested positively in AMES tests for possible carcinogenic activity. This remains a concern and there are, as well, a multitude of other ethical concerns associated with eradication strategies. As an example, it is still not known whether it will be facile to take patients, who are thought to potentially be cured of HIV, off of all antiretroviral drugs in order to test the hypothesis that eradication of virus might have taken place. Nonetheless, there is consensus in the field that ongoing research in this area is vital and that we must continue to make important progress in terms of having effective means of dealing with the HIV epidemic on a worldwide scale.
In Abstract 31, S. Palmer and colleagues presented their work on the characterization of persistent HIV-1 T cells isolated from blood and tried to correlate their findings with those obtained with tissue from patients undergoing long term antiretroviral therapy. Patients were evaluated who were acutely infected and compared with those who were chronically infected over periods between three and fifteen years. The goal of the study was to determine the sanctuary sites in which HIV persistence can occur. The results point clearly to the fact that both peripheral blood mononuclear cells, lymph node cells, bone marrow cells and gut associated lymphoid tissue can harbor HIV at various levels as sanctuary sites. Dr. Palmer's group enacted a phylogenetic comparison among these various cells to determine the potential of virus in these sites for evolution. The results document that the frequency of HIV infection in central memory T cells is higher among patients who started therapy during the chronic phase of infection in comparison with those who began therapy during acute infection. Similar results were obtained in regard to the other tissues that were studied, although bone marrow cells did not seem to be infected in a general sense, with the exception of CD4 cells located in this compartment. Dr. Palmer showed that there were 21 fold higher levels of viral DNA in the cells of patients who initiated therapy during the chronic phase of infection as opposed to the acute phase. This paper demonstrates the power of single genome and single pro-viral sequencing technics. Dr. Palmer did not have access to a series of longitudinal samples from the patients who were studied and thus is unable to estimate a precise time point at which a threshold barrier may be crossed in regard to numbers of copies of viral RNA and DNA that can be sequestered within latently infected cells.
Dr. Lewin also emphasized that Vorinostat will probably not be the best Hdac inhibitor to be employed in the context of efforts to eradicate virus. For one thing, this compound is somewhat toxic. Second, she emphasised that combination approaches will probably be necessary over the long term. She stated that other types of compounds that might also have applicability in regard to elimination of HIV reservoirs are methotrexate as well as ACE inhibitors. The ultimate goal is also to try to enhance delivery of these agents to all cells in the body, possibly through use of pro-drugs and/or nanoparticles. An additional important concept is that of targeting the myeloid lineage of cells. Dr. Lewin also mentioned the concept of using zing finger nucleases to attempt to eradicate CCR5 from the body's tissues as a means of forestalling future infections by HIV-1. Many questions of course remain, including possible dosing schedules for use of Hdac inhibitors. Questions also remain in regard to the safety of carrying out treatment interruptions and, as stated above, the ethics of withdrawing people from antiretroviral therapy.
In Abstract 32, GJ Besson et al studied the decay kinetics of plasma viremia in patients on antiretroviral therapy. Nine patients were evaluated in whom total amounts of HIV DNA were shown to decline after therapy was started. They also demonstrated that there some patients in whom HIV DNA declined relatively slowly and continuously and that these patients showed increases in the ratio of two LTR circles to total HIV DNA after initiation of therapy. However, the numbers of 2 LTR circles also subsequently declined. This study indicates that there may several different patterns of persistence and decline of sequestered viral DNA in different patient populations.
In Abstract 33, S. Fourati and colleagues described how HIV genomes can often be defective in infected cells after long term HIV therapy. They attribute this to the editing effect of Apobec3G and were able to demonstrate that viral genomes that harbored stop codons were significantly more divergent than those that did not possess stop codons a result attributed to the editing feature of Apobec. These investigators also observed that there was an inverse correlation between the size of the viral reservoir and levels of defective genomes within patients. They conclude that some reservoirs may include replication competent viruses whereas others may not. They further believe that comparisons of relative amounts of defective and non-defective viruses might be used as a criterion for the potential of achieving viral eradication in any individual and that measurements only of proviral HIV DNA might be insufficient in this regard.
In Abstract 35, G. Khoury et al demonstrated that naïve T-cells can remain extremely stable in terms of their ability to function as a reservoir for HIV, even beyond five years after initiation of antiretroviral therapy. They were able to demonstrate that memory T cells had significantly higher HIV DNA levels compared to naïve T cell populations. They further demonstrated that decays in levels of viral DNA were more rapid over the first twelve months following initiation of therapy compared to later time periods. Although naïve T-cells may be less problematic than memory T cells as stable reservoirs, they nonetheless may be recent thymic immigrants and may represent a very stable reservoir in the HIV infected patient population.
Abstract 36 by MF Kearney et al was on the topic of viral evolution in plasma during long term suppressive antiretroviral therapy. These workers assessed virus by single genome sequencing and were unable to document evidence of viral evolution at any time during exposure of patients to antiretroviral drugs. Nor were they able to demonstrate the presence of drug resistance mutations that might have been selected during ongoing therapy, suggesting that viral replication had not, in fact, taken place. Of course, this raises the subject of whether studies on viral evolution are adequate to determine whether or not low level viral replication may, in fact, occur in subjects who are subjected to anti-retroviral therapy. Indeed, it remains possible that low viral replication may not be accompanied by evolution given the small numbers of virus particles that are present, making it potentially difficult to detect meaningful changes. Another complication is the fact that certain drugs may not penetrate well into some sanctuary sites, thus potentially allowing low level viral replication in the absence of evolution, in the event that drug pressure was inadequate to select for resistant mutated forms of HIV.
Abstract 38 by RK Gupta et al was interesting in that it dealt with the importance of CD8 cell-mediated anti-viral immunity and the role that this plays in HIV replication and viremia. These authors described an elite controller who also suffers from cancer, yet, who has not had recurrence of HIV disease despite the fact that his malignancy has extensively compromised his immune system. The authors documented that this individual has high levels of CD8 cells and that these may be able to control HIV replication as shown by cytotoxic T cell assays and CD8-mediated interferon-γ responses. This work is important, because it highlights the important role that CD8 lymphocytes might be able to play in regard to limitation of HIV replication in elite controllers and, as well, that this seems to have occurred in the absence of high levels of neutralizing antibodies, perhaps because of anti-HIV CD8 activity.
Abstract 39 by SA Yukl et al was interesting because it deals with the so-called Berlin patient who has reported to have been cured of HIV infection. This group of investigators has conducted a series of exhaustive tests to determine whether HIV has truly been eradicated from this individual as a consequence of the bone marrow transplant that he received for therapy of acute myeloginous leukemia (from Jules: in reports emerging after this workshop it appears this investigation is trying to determine whether the Berlin Patient has a "sterilizing cure" or a "functional cure"). In addition, the investigators were interested in trying to define the potential role of various reservoir measurements in possible eradication. The results demonstrated that the patient remains ostensibly free of all live viral infection. This work was conducted on the basis of a large apheresis that yielded nine billion peripheral blood mononuclear cells that were evaluated for the presence of HIV-1. All of the cells tested were negative for p24 antigen, indicating that the frequency of replication was extremely low if indeed any residual live virus remained whatsoever. Several different laboratories have confirmed these results on this individual. In addition, the investigators reported that very low levels of HIV RNA were occasionally detected in the plasma of this individual. However, follow-up analysis of sequential samples indicated conflicting results; in addition, results for HIV DNA were always negative in regard to the cells that were tested. Although tissue derived from biopsies were occasionaly positive for very low levels of HIV DNA but not for viral RNA, these findings might suggest the presence of some residual viral non-infectious viral DNA in this individual. The results might therefore represent non-viable viral footprints that have been detected by ultrasensitive analysis. It is also noteworthy that antibodies against HIV in this individual were low and continuously to decline over the course of approximately 18 months. The investigators concluded that it is unlikely that this individual remains HIV infected and that it is not possible to conclude that HIV eradication has not occurred.
Finally, Abstract 37 by RF Schinazi and colleagues was on the topic of a novel series of inhibitors of HIV replication called Jak inhibitors. These compounds are able to interfere with the Jak-STAT pathway in activated lymphocytes and macrophages in the aftermath of HIV infection. Activation and reactivation of cells was performed using monoclonal antibodies against CD3 and/or CD28 and activation was monitored by flow cytometry. The Jak-STAT pathway is routinely stimulated in HIV-infected cells and can therefore be perceived as a logical target for drug development. Now, in this proof of concept study, it has been shown that two lead compounds termed Tofacitinib and Jakafi demonstrated excellent inhibition of HIV replication at concentrations ranging between 0.02-0.3 µM in cultured human lymphocytes and macrophages as well as against replication of a SHIV in lymphocytes and macrophages obtained from rhesus macaques. Moreover, these compounds were not toxic and the selectivity index for each of these compounds was high. This is the first demonstration that targeted blockage of the Jak-STAT pathway might be an effective way of interfering with HIV replication in both macrophages and lymphocytes. Moreover, the data suggest that these compounds might interfere with the ability of HIV to achieve latency in a variety of target cell types and that they were active against all of a variety of forms of drug-resistant HIV-1 that were tested. In addition, activity was demonstrated in regard to latently infected cells that were re-activated so as to become overt producers of HIV. The field will now await further studies on some of the molecules in this series, at least some of which are already approved by the Food and Drug Administration in regard to other disease indications, such as rheumatoid arthritis and myeleofibrosis.
Resistance to new antiretroviral compounds
Abstract 1 in this section was by D. Rajotte et al of Boehringer Ingelheim of Montreal, Canada. These investigators have developed a new series of nucleotide competing reverse transcriptase inhibitors that can reversibly inhibit the binding of an incoming nucleotide to the active site of reverse transcriptase. However, these compounds do not act as chain terminators. The objective of the current study was to evaluate drug resistance in regard to this novel class of HIV antagonists. The results have shown that these new drugs retain activity against viruses that include a wide variety of mutations associated with nucleoside compounds such as M184V and numerous thymidine associated mutations. Interestingly, there does not appear to be any cross-resistance to any of the non-nucleoside reverse transcriptase inhibitors such as efavirenz. In contrast, the presence of the K65R mutation seems to render these novel compounds 10-fold more active than they are against wild-type viruses. In tissue culture, these new compounds selected a novel W153L mutation that was present in 70% of clones and that was responsible for 210-fold full resistance against a lead candidate member of this new family of drugs termed compound C. At the same time, hypersensitivity on the part of viruses containing W153L was reported in regard to each of tenofovir, abacavir, 3TC, and FTC. Although some degree of toxicity seems to be present in regard to the members of this family developed until now, it is hoped that future drug development will lead to new products that are totally non-toxic and that will be able to enter clinical trials.
In Abstract 2, C. Hwang and colleagues of Bristol Myers Squibb reported on a novel compound termed BMS-986001. This compound, also termed 4'ED4T, is a novel thymidine analogue that is currently in phase IIb clinical trials. The results presented indicate that this new nucleoside maintains broad activity against a variety of viruses containing mutations associated with many other drugs. In particular, BMS-986001 was hypersusceptible in regard to the K65R mutation and also displayed good activity against viruses containing L74V. The addition of the M184V mutation, by contrast, restored viral susceptibility to only wild type levels. This novel compound also appears to be active against viruses containing only a few thymidine analogue mutations (TAMs) although an accumulation of TAMs led to a greater degree of drug resistance. In contrast, BMS-986001 also displayed hypersusceptibility against Q151M-containing viruses. In summary, this novel compound seems to have excellent potential in regard to becoming a future member of the HIV armamentarium of drugs. The field will look forward to seeing the data from the current phase IIb study and is hopeful that the further clinical development of this compound will occur.
In Abstract 3, M. E. Abram et al of Gilead Sciences in Foster City, California reported on resistance against Elvitagravir (EVG). It is well known that EVG is part of the so-called QUAD tablet that also includes cobisistat, Tenofovir, and FTC. The results of this study indicate that a variety of well-understood mutations can cause resistance against EVG and that most of these are also responsible for cross-resistance against Raltagravir (RAL). However, most of the mutated viruses that develop against EVG retained susceptibility to dolutegravir(DTG), the novel anti-integrase compound currently being developed by GSK. Indeed, the results of the study suggest that there is likely to be broad cross-resistance between EVG and RAL but that viruses that display resistance against either of these first generation integrase inhibitors may retain sensitivity against DTG. Further research will be necessary to investigate this issue and it would appear that the manufacturers of EVG wish to make the point that their integrase strand transfer inhibitor(INSTI), ie EVG, should be used in first-line therapy and that DTG might be used, if necessary, to salvage patients who might develop resistance against EVG in the rare cases in which this might occur. As will be seen below, the manufacturers of DTG have argued that their drug cannot always be used to salvage patients resistant to either RAL (from Jules: research suggests DTG can salvage many patients with RAL resistance) or EVG and that patients might be best served by beginning therapy on a DTG-based regimen. Presented at this workshop were the results of The VIKING study, which enrolled ART-experienced adults harboring RAL-resistant HIV. A broad range of RAL-resistant genotypes and DTG fold-change resistance were observed in the 24 Cohort II subjects. Through Week 24, 75% of subjects achieved <50 c/mL HIV-1 RNA, and 5/24 (21%) subjects experienced viral failure. The study required at least one fully active drug (PSS ≥1) in the optimized background regimen (OBR).- see this link to the NATAP report of this study -
(http://www.natap.org/2012/ResisWksp/ResisWksp_02.htm)
The ongoing phase 3 study hopefully will characterize which RAL-resistant patients respond to DTG.
In Abstract 4, K.L. White et al of Gilead Sciences reported on an analysis of drug resistance data that have emerged until now from the phase III QUAD studies that are likely to result in approval of Elvitagravir (EVG) within the next several months. An analysis of results obtained to date suggest that a group of mutations that are broadly associated with resistance against Raltagravir might also be associated with resistance against EVG. This notwithstanding, relatively few patients in the QUAD studies have developed resistance against EVG until now. Several who have failed on EVG have developed resistance against other components of the so-called QUAD regimen. 7 subjects who were in the efavirenz (EFV) arm of the phase 111 study that compared EFV against EVG developed the K103N mutation associated with resistance against EFV. Several also developed M184I/V mutations while very few (n=2) developed K65R, associated with resistance against tenofovir. In QUAD studies that evaluated individuals who received boosted atazanavir(ATV), EVG resistance was reported among several individuals but very little resistance against could be demonstrated. In general, these data highlight the promise of both the EVG arm of this study as well as both the other arms that have been evaluated that continue to show excellent antiviral activity. It now appears as though the use of boosted (ATV) in combination with TDF/FTC or EVG/cobisistat in association with TFV/FTC, and the original co-formulation of TFV/FTC/EFV all represent excellent options in regard to suppression of viral load in a context of once-daily dosing antiretroviral chemotherapy.
Abstract 5 was on the topic of the novel anti-integrase drug being developed by ViiV Pharmaceuticals termed Dolutegravir (DTG). This compound has shown excellent robustness in both pre-clinical studies as well as in a number of clinical trials performed to date. Now, results have been presented in regard to the Viking study that has evaluated the activity of DTG in combination with other drugs against viruses that were previously documented to have resistance against Raltagravir (RAL), the only approved member of the integrase strand transfer inhibitor family of drugs. The investigative team has studied a wide array of RAL-resistant viruses and have demonstrated that multiple subjects developed additional integrase resistance associated mutations during their time on DTG in this study. In some instances, these additional mutations have further reduced the sensitivity of viruses to DTG. The results cumulatively suggest that many patients who have initial resistance against RAL are unlikely to be able to benefit completely if later placed on a DTG-containing regimen (from Jules: down at the end of this report Wainberg says: "These findings highlight the fact that resistance against DTG may be possible to attain through accumulation of mutations and argue that DTG should best be used in first line therapy, even though this would not preclude its potential utility in second line regimens so long as the accumulation of mutations that confer resistance to RAL and EVG is not too far advanced"). These findings are important as they contradict to some extent findings from other groups that have suggested that full sequencing of INSTIs might be entertained as a possibility in the context of individuals who might fail therapy while on first generation INSTIs such as RAL and EVG. The current data also suggest that an initial Q148H mutation may be key in regard to subsequent non-responsiveness against DTG. Similar results were obtained regardless of level of dosing of DTG in the population of individuals studied.
Abstract 6 presented by M. Krystal of Bristol Myer Squibb was on the topic of a novel attachment inhibitor termed BMS-626529 that is being developed for antiretroviral therapy. This compound blocks attachment of HIV-1 to its CD4 receptor, by interfering with gp120/gp41 binding. A substitution at position M426L within gp120 was shown to be associated with reduced virological responsiveness to BMS-626529 although a different substitution at position S375M may also be associated with drug resistance. This notwithstanding, the use of BMS-626529 together with other excellent drugs may represent an excellent strategy toward suppression of HIV viral load. The presence of M426L seems to be, however, an excellent predictor of non-responsiveness against this compound. It is hoped that this compound will retain broad spectrum activity in regard to the pre-CD4 confirmation of the gp120-CD4 complex and will be developed for use in the clinic.
Mechanisms of HIV drug resistance
In Abstract 7, W. Sugiura and colleagues from Nagoya, Japan reported on a resistance analysis of the protease inhibitor darunavir(DRV). It is well-known that DRV is probably one of the best protease inhibitors (PIs) available at the current time. The results of this group have successfully demonstrated the emergence of DRV-resistant viruses through tissue culture selections. The resultant viruses harbor a wide array of well-known PI mutations and possess a protease confirmation that is distinct from that of wild-type protease(PR). These data, therefore, help to provide understanding into the rare instances in which resistance against DRV may occur.
Abstract 9 by K. A. Sutherland et al dealt with the role of mutations in the gag gene in regard to susceptibility and resistance against a variety of protease inhibitors. The results demonstrate significant difference in susceptibility against various PIs among viruses that are mutated in gag with up to 17-fold diminutions in PI susceptibility being reported in some cases. These results highlight the fact that significant differences in susceptibility to PIs can be based on mutations at gag cleavage sites. These findings further highlight the fact that mutational analysis of gag mutations is not currently being carried out, nor is this a topic that is being contemplated in regard to clinical usage in the near future. It should be pointed out that other studies, however, have reported very little impact of gag cleavage site mutations on clinical responsiveness. The field of gag mutagenesis in regard to susceptibility to protease inhibitors may be more easily demonstrated in molecular clones than in patient samples. According to some experts, this may reflect the results of recombination as opposed to actual mutagenesis of clinical significance within the viral genome.
Abstract 12 was presented by K. Hightower and colleagues in regard to results of the Viking study in which dolutegravir(DTG) has been evaluated in regard to activity against viruses that display resistance against raltagravir (RAL). These investigators have evaluated a number of mutations within the integrase gene that are associated with resistance against all members of the INSTI family of drugs. The results document that DTG retains a very long residency time in regard to the active site of integrase over protracted periods, despite the fact that major mutations such as those located at positions 148, 143 and 155 may be present. These data suggest that DTG can tolerate greater disruptions within the active site of integrase than can other members of the INSTI family of drugs such as RAL and EVG. Furthermore, these results highlight that an accumulation of multiple mutations associated with RAL and EVG may be required in order to cause diminished sensitivity against DTG. The results of the Viking trial, in which there have been a number of DTG failures, indicate that this can clearly be the case. The results also demonstrate that certain combinations of mutations associated with RAL such as G140S, Y143H, and Q148H can reduce the residency time of DTG to a half-life of 3.9 hours from more than 30 hours. Furthermore, the combination of E138A together with G140S, Q148H and N155H can reduce residency time to 30 minutes. These findings highlight the fact that resistance against DTG may be possible to attain through accumulation of mutations and argue that DTG should best be used in first line therapy, even though this would not preclude its potential utility in second line regimens so long as the accumulation of mutations that confer resistance to RAL and EVG is not too far advanced.
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