- is there a practically relevant decay of latent reservoirs?
- can strategies that activate latent cells further lower the viral load?
- can a combined approach lead to control of viral replication without drugs (functional cure)?
Apart from that, we must first find out how to approach the reservoirs methodically and what to measure.
Then he gave an overview of current ACTG trials:
- Planned study to look at the decay of reservoirs in different patient groups (acute / chronic infected, elite controller, blipper, patients failing therapy / resuppressing)
- A5281: studying a multi-antigen/cytokine/DNA-vaccine (already recruiting, so far no tolerability issues)
- A5286: Study looking at Rifaximin (an oral broad-band antibiotic that is not being resorbed and therefore active only in the gut). Can the microbial translocation and the resulting systemic inflammation be mitigated? Also the influence on the reservoirs is to be studied.
- A52301: Anti-PD1-Antibody: Quiescent cells harboring latent HIV can be forced out of latency with antibodies against the PD1-receptor and start producing virus. This study is still in concept phase.
- A52308: ART for elite controllers
- PR652: Effect of romidepsin (a cancer drug developed by Celgene that also acts as HDAC-inhibitor that can activate latent cells but isn‘t mutagenic in the Ames test as other drugs of this class) on the reservoirs: concept
Finally, Kuritzkes came back to the questions that need to be addressed:
Which endpoints should be looked at?- proof of biological activity on the target or the selected mechanism? - proviral DNA in PBMSs? - proviral DNA in cells of the compartments (which?)? - cell-/tissue associated viral RNA? - no viremia during analytical treatment interruption (ATI)
Here he pointed out the (dis-)advantages of an ATI:
-Best test for functional cure
-quantitative and qualitative analysis possible:
-Rebound of viral load (or lack of)- time to rebound - time to new setpoint - setpoint at a new (lower) level than before therapy
Cons:- Risk of inflammatory syndrome like during primary infection - risk for OI, CV-events, death (like in SMART-study) - increased transmission risk
We also have to think which patients are best being studied:
Finally, we must not forget the risk-benefit-balance since all new therapies will have to compete with established ART which is generally well tolerated and harbors usually only minor risks.
After that, Romas Geleziunas, Director of Virology at Gilead Sciences gave an overview of his company‘s activities in this area.
Gilead is focussing on the activation of HIV-expression in latently infected cells.
There are several possibilities:- De-repress chromatin (HDAC-inhibitors) - activate transcription factors (NF-ƘB) - activate HIV mRNA Elongation (PTEF-b) - Other mechanisms, still to be found by Hight-throughput analysis (HTS)
Gilead has developed an automatic system that can measure the reversal of latency with high numbers of samples at the same time. Already several new drugs have been discovered that can wake cells from latency. One of these, GSI-002 has the advantage of not being mutagenic and not activating T-cells itself. Some other compounds they found have such unbelievable names as "Thapsigargin“ or "Tyrphostin A“.
Another possible mechanism to eliminate HIV uses the "toll-like-receptor 7“ (TLR7). This receptor binds single-strand RNA and leads to the production of type-1 interferons (IFN alpha/beta). GS-9620 is a TLR7-agonist that has been shown to lower RNA and viral antigen production in several animal models. In a next step they will look whether this drug can play a role in eliminating HIV-infected cells. The mechanism of HIV latency needs to be understood still more fully and new drugs need to be discovered that play a role in or can interfere with this process.
Dale Ando, working for Sangamo Biosciences, once more presented the method of his company to knock out the gene for the CCR5-receptor with sequence specific zinc finger nucleases. This leads to CD4-cells that are immune against infection with CCR5-tropic HIV. Unfortunately, they didn‘t show any new data. The procedure seemed to have worked exceptionally well in one patient, who is heterozygous for the Δ32-mutation, i.e. he has only one functional CCR5-gene per cell. Therefore, the next studies will recruit preferentally such patients. Furthermore, the procedure will be adapted for stem cells. The problem is, that the introduction of the zinc finger nuclease into the cells also triggers the differentiation process and the cells wouldn‘t be stem cells any more. Sometimes the devil is lurking in the details... Good news is, that the procedure has been automated, so more patients can be treated which facilitates larger studies.
Birger Sørensen from Bionor Pharma, a small Norwegian Biotech company with only 19 employees presented a very interesting approach: They changed certain highly conservated regions of the p24 protein in a way that makes it much more effectively recognized by human immune cells. For some time it was already known that infected people with a strong immune response against p24 have a slower disease progression. In order to further strengthen the vaccine response, they use their product called Vacc-4x together with GM-CSF (a growth factor for granulocytes/macrophages). Four primary and booster immunizations in 40 patients led to 92% showing a immune response with good tolerability. In another study patients were immunized while on ART, then there was a 10 week waiting period to allow the immune system to calm down and finally ART was stopped. In all patients the viral load rebounded upon interruption, but the vaccinated patients reached a new setpoint that was about 0,4 log lower (i.e. average viral load 20.000 cp/ml instead of 60.000). They hope to further lower the setpoint with more round of vaccination. In addition they try to improve the immune response with immune modulators like lenalidomide.
Another approach, called Vacc-C5 aims in a different direction: antibodies against the C5-region of gp120 cannot neutralize the virus, but they still lead to a slower disease progression. This is probably because the complex of C5 and gp41 bears some similiarity with the human HLA-complex and can hyperactivate the immune system. Antibodies can block this hyperactivation and so slow down the disease progression. In animal trials it was possible to produce antibodies with the Vacc-C5 vaccine. Trials in humans are planned. Both approaches together would make use of the cellular as well as the humoral arm of the immune system and such a combined approach could be an important step on the way to a cure. The main problem right now is money because studies are expensive and a small company like Bionor is heavily dependent on investors.
John Zaia from Beckmann Research Institute gave an overview over the state of stem cell research in the area of HIV. It was a stem cell transplant that led to the cure of the "Berlin patient" and brought cure research back on the scientific agenda of the HIV researcher. But it is still unknown which factors were important for this experiment to succeed. Some researchers think that the graft-vs.-host-reaction, which almost killed the patient, was crucial - in addition to the radiation and the intense chemotherapy. So far, there is no second "Berlin patient", probably because the combination of the right tissue antigens and the CCR5-Mutation is rather rare. That‘s why they try to introduce the required CCR5-mutation artifically in bone marrow cells of donors. Another possibility to get stem cells is cord blod from newborns. But again here is the problem of the rare occurence of a CCR5-Mutation so there are plans to found a blood bank of cord blood with CCR5-Mutation. Alltogether the field of stem cell therapy is still in it‘s infancy. In patients who don‘t need a stem cell transplant for medical reasons (i.e. lymphoma), the risks are still too high. This method has only a chance for broader use when there will be new developments making radiation and chemotherapy unnecessary.
Pablo Tebas gave an overview over the activities of his workgroup. Apart from collaboration with Sangamon in the field of zinc finger nuclease (see above), the group also tries to increase the CD8-mediated killing of HIV-infected cells by providing the CD8s with a new T-cell-receptor that can recognize HIV much better than it‘s natural counterpart. Clinical studies including 16 weeks of ATI and rectum biopsies are planned. And there is renewed interest in the long known antiviral activity of interferon alpha against HIV. In a study with HIV patients that had undetectable viral load, stopped therapy and used pegylated interferon alpha as monotherapy, the viral load could be kept below 400 cp/ml in 45%. At the same time, the number of integrated HIV genomes in the circulating CD4-cells went down. Interestingly, this effect was also seen in a group of patients receiving only half of the standard dose (90 µg instead of 180 µg interferon alpha per week) - with much better tolerability.
David Evans and Nelson Vergel, two of the activists who had organized the workshop, did a internet survey about the willingness of patients to participate in clinical studies that are not of immediate benefit to them but maybe even have some serious risks. They found among many other things, that unexpectedly many patients would consider participating in such studies for mainly altruistic reasons.
Steven Deeks provide additional insight in the mechanisms of viral persistence of HIV:
Deeks described these mechanisms in further detail and also some ideas how to overcome them, e.g. antibodies against PD1 to reverse latency.
Keith Jerome and Hans-Peter Kiem were the last presenter and described their efforts to
For the first goal they also use zinc finger nucleases. The viral reservoirs will be attacked with different tools: One idea is to mark infected cells with siRNA-probes to make them visible for the immune system so that killer cells can get rid off the infected cells. Another method uses "homing endonucleases", special restriction enzymes from yeast. The enzyme Y2-Anil has been changed in a way to recognize HIV-specific areas in the genome and cut them. These cuts will be recognized by the cells repair system and the gaps will be filled with random nucleosides. This leads to "nonsense“ viral sequence that doesn‘t produce active virus anymore. This method has a big advantage against the Tre-recombinase that has been described some time ago. Y2-Anil recognizes longer, very conserved regions of HIV so that it could be effective against a broad range of different HIV types whereas Tre recombinase is limited to the LTR region of HIV (in fact the efficacy of Tre recombinase has been shown only against a very artificial laboratory strain of HIV and not the wild type virus).
All speakers of this exceptional workshop shared the view that we must not raise premature and unrealistic hopes in patients. First steps have been made but we need many more successes in analytics, basic science and translational research in the animal model as well as in patients before the cure will be within reach.