Immediately prior to the main IAS conference there was a two-day meeting with an excellent programme focusing on the growing body of research looking at targeting the HIV viral reservoirs, both tissue compartments and latently infected cells. The reports below are rappporteur summaries from the IAS website. Unfortunately this meeting was not webcast, however, many of the powerpoint presentations are posted online.
Where the posters from the meeting were also presented to the main conference, these abstracts are also online.
Steve Deeks from the University of California San Francisco addressed two major questions: the clinical implication of residual viraemia on the pathogenesis of non-related AIDS diseases and how inflammatory mechanisms and T-cell activation can contribute to viral persistence.
Despite the strong impact of ART on survival and quality of life of HIV-infected patients, a gap in expectancy of life between non HIV-infected people and ART-treated patients still remains. Actually although HAART improves vascular function it does not completely restore vascular health (Hsue et al. CROI 2010). How could we explain this picture?
Two consequences of HIV infection persists in treated patients: residual viraemia and inflammation markers. The question to answer is whether both events are related to each other, and more importantly, if this impacts on mortality due to non-AIDS events, in particular cardiovascular diseases, in ART-treated patients.
Little evidence supports a direct role for residual replication as a factor involved in both inflammation and non-AIDS related diseases. Cohort studies and most intensification treatment trials have failed to show a relationship between residual viraemia and inflammatory parameters (Dinoso et al. PNAS 2009, Hatano et al. CROI 2010).
An inflammatory state represents a risk factor for the development of cardiovascular disease. In this context, numerous studies have shown persistent T-cell activation and increased inflammatory markers in chronic HIV infection but also in ART-treated patients. Increased immune activation parallels a lack of recovery of vascular health (Kaplan et al. CROI 2010). A best knowledge of the real impact of different factors causing chronic inflammation such as continuous bacterial translocation, CMV viremia, increased levels of cytokines and D-dimer (Volderbing and Deeks, Lancet 2010) represents a central issue of research.
Regarding the mechanisms of viral persistence in reservoirs and in particular the potential role of the immune system in the maintenance of such reservoirs different authors have shown a close relationship between immune activation levels and the size of the reservoir. Particularly relevant is the size of the reservoir in the gut that increases the number of infected cells tenfold in comparison with classical estimates (Yuki et al JID 2010). Strong activation of the GALT system undoubtedly contributes to higher susceptibility to infection of lymphoid cells in this compartment. Together with these mechanisms, homeostatic proliferation of particular subsets of memory T cells further contributes to expansion and persistence of the latent reservoir (Chomon et al, Nat Med 2009).
As a general conclusion, in the ART era we face a new scenario in which the wild destruction of the immune system and lethal opportunistic infections have been replaced by new mechanisms of immune dysfunction and new causes of morbidity in HIV-positive patients. These non-AIDS events probably are not related with persistent residual viraemia but with chronic activation and premature senescence of the immune system. To understand the pathogenic mechanisms of this new scenario represents a major challenge in order to improve the health of patients and to reduce the survival gap still remaining in comparison with HIV-negative people.
Session 1: Where and what are viral reservoirs? HIV reservoirs and sanctuary sites
Satya Dandekar opened the session pointing to the major questions to be addressed in the field of viral reservoirs. Two levels in assessing viral reservoirs are usually considered, cell types and anatomic tissues. A spectrum of viral reservoirs must be considered because cellular types differ in their capacity to sustain productive or latent infection and their susceptibility to viral cytopathic effect. Regarding anatomical reservoirs, gut and brain are probably the main tissues hosting HIV reservoirs during ART and interestingly both sites share two characteristics: the presence of activated cells and a microenvironment that can make drug accessibility difficult.
Among others, the following questions were raised: the potential role of early treatment to reduce both the size of the reservoirs and to allow better immune reconstitution of the GALT system; the impact of ART intensification to reduce reservoir size; and, the role of natural immunity to control HIV reservoirs. Finally the relevance of SIV models to answer these questions was also emphasised.
Three presentations addressed the role of gut as reservoir in ART treated patients and two communications focused on the origin of viral reservoirs in CNS.
Data assessing CD4 and lymphocyte activation in different gut regions were discussed by Steven Yukl from the Plus study group. Previous work presented at CROI 2010 by this team showed that levels of HIV DNA in CD4 T-cells varies across gut sites and suggested that the terminal ileum could represent an important reservoir site and a source of ongoing replication despite ART. To get better insight into the role of the ileum in HIV infection, sample biopsies from 10 HIV-positive people on ART and 8 HIV-negative controls were compared. CD4 levels were lower in ileum from HIV-positive people despite ART, but approached to normal recovery in rectum. Interestingly, a different distribution of memory subsets between blood and gut and among gut sites was found. A decreased proportion of effector and transitional memory cells was found in ileum and these differences could account for the higher HIV DNA content in CD4 T cells in gut. Although these data should be confirmed in a large number of patients they suggest the existence of different compartments in the gut regarding cellular distribution, response to ART and reservoir size.
Gabriella dEttorre from University of Rome analysed intestinal biopsies from sigmoid colon in either 14 ART-treated or treatment-naive patients, to correlate HIV-DNA levels, immune activation and microbial translocation. They found a 50% reduction in proviral HIV DNA levels in treated compared to naive patients, but very incomplete reduction of microbial translocation and T cell activation (<20%) was found between both groups. HIV-DNA level in the GALT correlated directly with the levels of LPS suggesting that mucosal damage and the size of the reservoir directly contribute to chronic immune
activation in ART-treated patients.
Finally, John Zaunders from St Vincents Hospital at Sydney, analysed HIV-1 DNA levels in different lymphocytic subsets from eight HIV-positive patients with high CD4 counts. This analysis focused on memory CD4 displaying the integrin pattern a4+b7+ that define gut-homing CD4 T cells, that can migrate from blood into GALT and back into blood via draining lymphatics and thoracic duct. The authors proposed the hypothesis that these gut-homing cells should be preferentially infected but their results clearly show that the majority of HIV DNA was found in CD4+ T cells that were not gut-homing (b7-). Therefore, these provocative data suggests that the majority of infected CD4+ T cells in PBMC were unlikely to have been activated and infected in GALT. The question remaining is where they become infected?
Two presentations addressed the role of viral replication in CNS. Melissa Churchill from Burnet Institute in Melbourne studied the infection of astrocytes in the brain of 14 patients with different degrees of HIV-associated encephalitis (HIVE) and HIV-associated dementia (HAD). Combining laser capture and single-cell microdisecction with sensitive Alu-PCR, integrated HIV-DNA was found in astrocytes. Two aspects are remarkable in the work: first that the magnitude of astrocyte infection that varies between 0 and 20% correlates with HAD and severity of HIVE. Second, that astrocyte infection frequency correlates with proximity to macrophages and is maximal at the perivascular regions of the deep white matter suggesting the transmission by trans-migrating cells. Further studies analysed the activity of the HIV-LTR isolated from infected astrocytes that displayed lower activity than LTR from PBLs in the same patient but transcriptional activity was triggered by HDAC.
Finally, Ronald Swanstrom from University of Carolina at Chapel Hill, through in depth sequence comparison between blood plasma and CSF, showed a compartmentalisation that was associated with neurological status. Whereas in asymptomatic subjects similar sequences were found in blood and CSF, in HIV-associated dementia (HAD) specific sequences were exclusively found in CSF, strongly suggesting active viral replication in the CNS compartment. Based on the decay rate of the virus in CSF in patients with viral encephalitis, either T cells or macrophages were proposed as sources of viral replication. In patients with viral compartmentalisation and fast decay following ART, HIV would replicate in brain T cells whereas in those patients with slow viral decay in CSF the source of virus would be macrophages or microglial cells. Phenotypic data trying to correlate this different kinetics with infection of different cell lines were discussed in support of this hypothesis.
Overall, both presentations showed consistent and provocative data that highlights the role of the CNS as a viral reservoir that needs to be considered when designing strategies aimed at a cure of HIV-1 infected individuals.
In summary, this session addressed particularly relevant issues and probably more questions than answers were generated. Particularly relevant was in many presentations the use of new technologies to analyse and quantify the reservoir size in gut and CNS in a precise manner. However, the extreme difficulty to get material from gut and CNS represent a major limitation in the study of such still hidden reservoirs in vivo.
Session 2: What are the mechanisms of persistence?
This session was dedicated to discuss the molecular mechanisms involved in the establishment and the maintenance of latent HIV reservoirs and its dynamic nature.
First, Dr Eric Verdin in his overview talk focused on HIV transcriptional silencing leading to the establishment and maintenance of latency. Work from different laboratories suggests that transcriptional silencing of HIV is a multifactorial process that involves: lack of key transcription factors (e.g. NF-kB, NF-AT, STAT5, P-TEFb); recruitment of chromatin modifiers (e.g. HDACs, Suv39H1); and DNA methylation. All of which lead to the formation of a repressive chromatin environment. Three talks mainly dedicated to the molecular mechanisms of transcriptional latency followed.
First, Dr Alessandro Marcello explored the possible correlation between nuclear localisation and HIV transcriptional status. In the latency model used HIV was found in the nuclear periphery. Activation of the provirus did not change its localisation. Reports from different laboratories show that in its transcriptionally repressed state, the viral promoter is stalled due to the action of negative transcription elongation factors. Positive transcription elongation factor b (P-TEFb) is a critical cofactor for the viral protein Tat which is required for efficient elongation of HIV mRNA. In his effort tounderstand the molecular mechanism involved in CTIP2-mediated repression of HIV promoter, Dr Rohr found that CTIP2 exists in 2 repressive complexes. One containing HDAC2 and the methyltransferase SUV39H1 while the other is composed of CTIP2 and inactive PTEFb. Thus, CTIP2-mediated repression of P-TEFb activity contributes to HIV latency.
Dr Bijan Sobhian then characterised the composition of Tat/P-TEFb complexes. He found that Tat exists in two biochemically distinct complexes. The active complex, Tatcom1 is composed of core PTEFb and additional new factors known to play a role in transcription elongation. This multifunctional complex contains proteins important for optimal PTEFb activity. Tatcom1 subunits are recruited to the viral promoter in Tat dependent manner. Knockdown of this subunit reduced Tat transcriptional activity. Thus, active P-TEFb should be stimulated to reactivate HIV from latency. An important challenge for the future is to find a way to target active PTEFb to silence HIV promoter.
Dr Tae-Wook Chun emphasised on the dynamic nature of the latent reservoir. Low-level viraemia exists in a majority of HAART-treated patients and is not well understood. Ongoing viral replication may account for this viraemia in some patients. Understanding where this virus comes from will be essential to purge the latent reservoir.
Finally, Dr Gero Hütter led a discussion about the recent case in which a delta CCR5 bone marrow transplant allowed a patient in Berlin to stop HAART without any rebound in viremia. HIV RNA and DNA were undetectable in patient samples. These results suggest that reconstitution of the immune system by CCR5 negative bone marrow also eradicates the latent reservoir. [Editorial note: or that the reservoir persists but new virus is unable to reestablish infection in CD4 compartment due inability to enter these cells].
In conclusion, this session highlighted the multifactorial nature of HIV latency and the long way we still have to go to understand it fully. Combinatorial therapies or identification of common mechanism will then probably be required to reduce the latent reservoir in HAART treated patients. Given the paucity of latently infected cells available (1/106 lymphocytes), it is also difficult to work on primary cells from patients. Since the different models of latency have many shortfalls, isolating or at least enriching the population of latently infected cells obtained from patients is a key issue.
Session 3. What is the role of the immune system in HIV persistence?
Brigitte Autran provided an overview for the session, highlighting the good and bad roles of the immune response to HIV-1 infection. Also highlighted was the potential of exhausting the HIV-1 reservoir through a combination of therapy intensification and immune interventions such as IL-7 or HDAC inhibitors.
Central memory CD4+ T-cells (TCM) and transitional memory CD4+ T-cells (TTM) represent the major reservoir of viral DNA. It is possible that through IL-7 triggered homeostatic proliferation (the ability of T-cells to divide in the absence of activation), the latent reservoir could be expanded. Vicente Planelles and colleagues described a system where TCM cells were generated ex vivo. When infected, these cells were shown to have a high proportion of integrated virus but a low proportion of productive infection. IL-7 was shown to be a poor reactivator (1/8 efficiency of µCD3/µCD28) of latent virus. IL-7 could however, cause vigorous proliferation of TCM cells. The authors concluded that IL-7 induced cell division could occur with minimal viral reactivation, suggesting that homeostatic proliferation could be expanding the latent reservoir.
The depletion of CD4+ T-cells in the gastrointestinal associated lymphoid tissue (GALT) leads to increased microbial translocation, which is proposed to contribute to systemic immune activation. David Asmuth and colleagues sought to determine whether CD4+ T-cell depletion in the GALT and systemic immune activation correlated with the presence of pro-inflammatory gram-negative bacteria orders in the gut. Presenting a pilot study of individuals pre- and post-initiation of therapy, higher proportions of pro-inflammatory gram-negative bacterial orders were associated with duodenal CD4+ T-cell depletion and systemic immune activation. This suggests an immunopathogenic role of bacteria in the duodenal tissue of HIV-1 infected individuals.
Resting CD4+ T-cells are a reservoir of latent infection. Vanessa Evans et al. investigated whether dendritic cells (DCs) played a role in the establishment of a latent infection in resting CD4+ T-cells. Using an in vitro model of HIV-1 latency in resting CD4+ T-cells, co-culture with myeloid dendritic cells (mDCs) induced a latent infection. This promotion of latency required both cell-cell contact and soluble factors. The authors suggest a possible pathway for the establishment of latency in vivo in lymphoid tissues.
The HLA-B27 and HLA-B57 alleles are over-represented in Long Term Non Progressors (LTNPs). Benjamin Descours and colleagues sought to investigate the impact on HIV-1 reservoirs by the strong immune control mediated by these protective alleles. Using a cohort of LTNPs, the B27/B57 alleles were associated with lower infection levels in TCM cells. Anti-gag CD8 T-cells were associated with increased TCM pool size. Thus the authors concluded that strong HIV-specific immunity in HLA-B27/57 patients results in maintenance of a larger and healthier TCM pool.
HIV-1 elite controllers (ECs) are a small proportion of HIV-1 infected individuals who have undetectable viraemia in the absence of therapy. The role of immune protection in these individuals is not well defined. In this light, Mathias Lichterfeld and colleagues assessed the role of p21, a host protein shown to inhibit HIV-1 replication, in elite controllers. CD4+ T-cells from ECs were less susceptible to HIV-1 infection and these cells had significantly higher expression of p21. p21 acted by reducing both HIV-1 reverse transcription and mRNA transcription. This study demonstrated that p21 up-regulation in ECs is part of the immune control of HIV-1.
Session 4: What host factors are at a play?
Host factors influencing HIV-1 infection and/or pathogenicity offer an attractive opportunity for therapeutical intervention. However, only a handful of such factors have been shown to be clearly relevant so far. Paul de Bakker overviewed recent Genome Wide Association Studies (GWAS) that aimed to identify host genetic differences influencing HIV viral load (VL) or disease progression. All GWAS clearly showed that variants in CCR5/CCR2 and in the Major Histocompatibility Complex (MHC) influence both parameters. However, this has been known for a few years, and although some new candidates outside the MHC have been proposed to modulate VL, the associations with VL are lost when larger samples of datasets are analyzed. Thus, Paul de Bakker assumes that GWAS have pretty much failed so far in providing robust associations of new gene variants influencing HIV-1. Larger cohorts of individuals or alternative approaches, such as whole genome sequencing or the integration of more clinical parameters may be needed to obtain more significant information, especially in the case of rare variants.
In the meantime, significant advances have been achieved in the understanding of the mechanisms of action of classical host factors which impact different steps in the replication cycle of HIV-1. RhTrim5-alpha blocks HIV-1 replication by interacting with the capsid after viral entry hindering completion of reverse transcription. The inhibition of the cellular proteasome rescues reverse transcription while maintaining the block of infection, suggesting a role of the proteasome in the blockage. Cindy Danielson showed that HIV-1 infection induces the relocalisation of the proteasome from the nucleus to rhTRIM5-alpha-containing cytoplasmic bodies. Danielson was able to visualise long term associations of the proteasome with flurorescent viruses moving dynamically through the cell, which often resulted in a decrease in the virus signal. Although it has been clearly demonstrated that the cellular factor LEDGF/p75 has a deep impact in HIV-1 replication by interacting with integrase and targeting viral integration towards active transcription units, it remained controversial whether LEDGF/p75 is essential or not for HIV-1 replication.
Rik Luk Guy Schrijvers reported the analysis of the first LEDGF/p75 integrase binding domain knockout human cell line. LEDGF/p75 knockout does not avoid HIV-1 replication although it results in the inhibition of single round infections and much delayed replication of productive virus. The number of integrated copies is reduced in LEDGF/p75 knockout, and integration in these cells occurs away of genes and preferentially in CpG islands. Once the virus integrated, some host micro RNA (miRNA) can modulate HIV-1 expression contributing to virus latency.
By performing miRNA qPCR array analyses of CD4+ T cells from HIV-1 exposed uninfected individuals (EU), elite long term non progressors with undetectable viral load (eLTNP), viremic patients and healthy donors, Claudio Casoli showed that the first three groups of individuals shared an altered expression of 8 miRNA, when compared to healthy donors, which may be interpreted as a signature of exposition to the virus. Furthermore, another pattern of expression allows distinguishing infected individuals (both eLTNP or viremics) from EU, which happen to have significantly lower levels of DICER and DROSHA, enzymes involved in the processing of miRNA. Exposition of CD4+ T cell to recombinant gp120 induces the same alterations in the patters of miRNA expression observed in vivo, which suggests that gp120-CD4 interactions may be responsible of the modulation of miRNAs. For leaving the infected cell, HIV-1 needs to overcome, via the viral protein Vpu, the effect of the restriction factor tetherin, which tethers virus to the cell surface inhibiting virus release.
Because, theterin localises into lipid rafts in the cell membrane and rafts are involved in the establishment of the viral synapse and HIV-1 cell-to-cell transmission, Bjørn Kuhl hypothesised that tetherin might affect this mechanism. Using transduced cells lines expressing or not tetherin, Kuhl observed that when tetherin is present in the cell surface it inhibits cell-to-cell transmission of HIV-1 devoid of Vpu, confirming recent observations by other groups. However, this effect of tetherin is also counteracted by Vpu. Interestingly, in the absence of tetherin, the transmission of the Vpu mutant was better than that of the wild-type virus, suggesting that Vpu may present a fitness cost in cell-to-cell transmission that is outweighed by the advantage of anti-tetherin action.
Session 5: What are the potential therapeutic interventions and how to evaluate them?
The session was started by co-chairperson, Christine Katlama, who said that the thought of a cure for HIV should be freed from the realm of science fiction in light of novel results.
Frank Maldarelli then addressed the need of precise quantitative measures for ongoing viral replication during antiretroviral therapy (ART). In his view, residual viraemia during ART does not depend on the potency of antiretroviral drugs, and intensification of ART with NNRTIs or protease inhibitors does not contribute to abatement of residual viremia. He however quoted a recently published study (Buzón et al., Nat Med 2010), in which some patients with undetectable viraemia did show evidence for further antiretroviral effects upon ART intensification with integrase inhibitor raltegravir.
In this regard, Una O’Doherthy, used a novel assay for the measurement of residual viral replication, and showed that two thirds of patients on ART sporadically had an excess of non-integrated proviral DNA, strongly suggesting that viral replication cycles
sometimes occur despite ART.
The presentation by Carolina Garrido showed that switching an ART regimen to a raltegravir-containing regimen conferred additional immunological benefits such as increases in CD4 counts, which might be caused by an increased thymopoiesis.
Dr. Carolina Gutierrez presented results from ART intensification using the CCR5 inhibitor maraviroc. She obtained a decrease in the numbers of cells infected with replication-competent HIV-1 in peripheral blood and a decrease in the activation levels of CD4+ and CD8+ T cells at the long term. Novel strategies aimed at curing HIV-1 were also presented in this session.
As pointed by co-chair Alain Lafeuillade, these can be distinguished in strategies reverting HIV-1 latency and in the so-called viral “sabotage” strategies, reducing the capacity of the viral reservoir to expand.
Among the former strategies, Sandrina Da Fonseca presented in-vitro data showing that CD4+ T cells expressing high levels of the surface antigen PD1 from HIV-1 infected patients are enriched in integrated and total HIV-1 DNA and expression of PD-1
correlated with the size of the viral reservoir. She showed that this may be due to the fact that PD-1, by interacting with its ligand PD-L1, maintains proviral DNA in a latent status. Disruption of this protein-protein interaction through drugs or antibodies induced viral replication of integrated provirus, which might represent a novel therapeutic avenue.
As a sabotage strategy, Dr. Andrea Savarino presented his latest in-vivo data using the gold-based compound auranofin (Gar1041). This drug induces down regulation of the cell survival-associated antigen CD27 in central memory T-cells, likely decreasing the
half-life of this reservoir for the latent provirus. When administered to monkeys infected with SIVmac251 and treated with intensified ART, a significant decay in proviral DNA was observed, as well as a delay in the rebound of viraemia following therapy suspension. Moreover, following this treatment, monkeys acquired an ability to maintain low-level viraemia and high CD4 counts in the absence of ART, thus opening new avenues for obtaining a drug-free remission of the infection.
Sharon Lewin highlighted social issues linked to the importance of research into a cure for HIV. Full life expectancy is not restored by ART, according to data derived from the Danish HIV cohort and showing that HIV-positive individuals may to date expect to live a life 70% shorter than the healthy. Even this reduced life expectancy is not accessible to each of the people living with HIV in the world: for every two people starting ART, there are five new HIV infections, and the total projected economic resources required to control the disease are increasing.
As shown by the important results obtained from the beginning of the epidemic (diagnostic tools, ART), community engagement will be important for reaching the ambitious goal of eradicating HIV. Basic science should indeed meet the requirements of people living with HIV (PLWH). One example is that methods for quantifying HIV reservoirs, such as lymph node and gut biopsies are highly invasive. Moreover, clinical trials for strategies aimed at HIV eradication are currently needed by the PLWH community. Finally, a higher level of involvement of politicians and media would be important for addressing resources to this ambitious goal.
Daria Hazuda highlighted the difficulties in the drug discovery process. From 5,000–10,000 compounds (but these numbers may be higher) that are synthesised for in-vitro evaluations, approximately 250 are selected for further preclinical investigations
in cellular and animal models. Among these, an average of five compounds is addressed to clinical trials and only one of them obtains FDA-approval. In this context, the research for drugs aimed at curing HIV/AIDS offers special difficulties. In the case of
antilatency compounds, there are several cellular models available (LTR-reporter constructs, chronically infected, inducible cell lines, retroviral vectors, and primary cell models), and the results obtained in these different models may not be comparable. Merck has put major effort in the development of histone deacetylase (HDAC) inhibitors as antilatency drugs. HDAC inhibitors could be used in combinatorial approaches, as they act downstream from the “sparking signal” required for activation of HIV-1 transcription (provided by nuclear factors). In this regard, protein kinase C (PKC) activators may play a major role in providing the “sparking signal”, and some of them are synergistic in combination with HDAC inhibitors in inducing HIV-1 escape from latency in vitro. When HDAC inbibitor, vorinostat and a PKC activator were administered to a monkey model for lentiviral (SHIV-RT) persistence during ART, it was possible to show decreases in tissue viral DNA levels but no delay in viral load rebounds when therapy was suspended. This result may however be improved by combining this approach with other strategies.
The session was closed by Paula Munderi, highlighting the need of controlling HIV spread in serodiscordant couples, and Christine Rouzioux, who addressed the need of studying those rare patients showing a remission of the disease after therapy suspension.
Rapporteurs: José Alcamí (opening and S1), Xavier Contreras (S2), Michael Roche (S3), Asier Sáez-Cirión (S4), Andrea Savarino (S5 and closing).