7 HIV Persistence Workshop: second day
The 7th International Workshop on HIV Persistance during Therapy started on Tuesday December 8.
276 participitants are present and the sessions are very interactive.
Today, we will deal with "In vitro and in vivo models of HIV persistence", "Basic science of HIV latency", "Virology of HIV persistence" and "Anatomic and non-CD4 cell reservoirs".
Finally this second day will end with a 1:30 hour of poster viewing session.
SESSION 1: In vitro and in vivo models of HIV latency
Victor Garcia Martinez (Chapel Hill) presented the different humanized mouse models that are available.
Two models are currently used. One is the bone marrow (HSC) thymus/liver transplant (BLT), the otheris the MoM model where only Bcells and myéloid cells are engrafted.
In the BLT model it has been demonstrated that a combination of antibodies can reduce viremia and that the addition of inducers (SAHA, I-BET, ...) can lead to no rebound.
The advantage is that viral outgrouth assay can look at all tissues of the animal.
The 3B3-PE38 immunogen, an immunotoxin, has been given to animals on ART and provided a 10 fold reduction in RNA present in T cells. Furthermore, the toxin killed the cells.
The MoM model (myeloid only mouse) was infected by different HIV strains and no growth was found. It is only when a chimera with HIV isolated from a patient with cnS complication was used that viral growth developped in MoM.
All tissues were positive in this MoM model with the viral outgrouwth assay.
The infected macrophages were able to migrate to the brain of MoM.
Dr Hughes (Frederick) studied clones of SIV infecting macaques. Some clones are tissue restricted (lymph nodes, spleen), others not. In fact, in most cases, 50-70% of the virus is provided by a single clone.
Dr Marsden (Los Angeles) studied latency reversal by PKC modulators in the BLT mice.
There are large catalogs of PKC modulators. These compounds induce CD69 expression in primary CD4+ T cells.
He used a NL-HA strain which is X4 tropic and infected BLT mice. He found a dramatic loss of cell viability after in vivo activation in the presence of PKC modulators.
Dr Woelk (San Diego) used the Planelles model v2.0 of HIV latency to compare gene expression and micro RNA signaling pathways between latently infected cells and those who are not.
Dr Aldovin (Boston)described in vivo suppression of SIV-mediated immune activation by a P38 MA PK inhibitor combined to ART.
P38 MA PK inhibitos have been developped for several conditions. He used the Pfizer PH-797804.
In the presence of the inhibitor alone, nothing happened on viremia.
There is a limited but significant greater increase of the percentage of TCM cells in the ART + inhibitor group of 8 animals.
The inhibitor improved the relative balance of PBMC Th17/Treg CD4+ subsets. It decreased immune activation of about 25-30% in PBMC and also in rectal biopsies.
Dr Kupla (Frederick) gave an elegant presentation.
They have developped a model of HIV platency which is physiologically relevant, memory subset driven, scalable and stable over time. It is called LARA (Latency and Reversion Assay).
LARA in vitro simulates the contribution of the different CD4 T cell subsets to the reservoir (TCM > TTM >TEM) and can be used to test an agent on only one subset.
With most latency reactivity agents, LARA shows inefficient latency reversal in TCM although it contributes mostly to the reservoir.
Dr Petterson (Seattle) quatified the impact of autologous transplantation on viral reservoirs in pigtail macaques. Actually, he showed that rebound in SIV viremia is higher in transplanted animals after ART is stopped (pic and set point) which raises 2 hypothesis:
-either the reservoir expands during ART
-or TBI potentiates viral rebound.
In non transplanted animals, lymph nodes and lower GI tract sites expressed viral RNA during suppressive ART. In the transplanted animals, all the tissues expressed viral RNA, even when they are still suppressed by ART in the periphery.
SESSION 2:Basic science of HIV latency
Jonathan Karn (Cleveland) gave a talk on epigenetics.
He showed that the polucomb PCR-2 is required to both establish and maintain latency.
He also showed that E2H2 inhibitors reactivate latent HIV-1 in Th17 primary cells.
Silenced populations are heterogenous for H3K23me3.
JARID 2 and IncRNA are probably required for HIV silencing.
Epigenic silencing of HIV is, in fact, progressive and hierarchical.
Dr Ciuffi (Lausanne) used Fluidigm technology to analyze cells individually.
Most of the highly permissive cells appear to be activated and cells which are low permissive are less activated.
She studied 7 different biomarkers of cell heterogeneity and found that CD62+/Tim3+/CD40L+ best characterize permissivity, even within the CD25+ activated population.
Dr Cillo (Pittsburgh) showed that cell-associated (CA) HIV RNA and CA HIV DNA are strongly correlated and both correlates with spontaneous virions production in cultures from resting CD4 cells.
Dr Romerio (Baltimore) talked about antisense transcripts (AST).
AST are inducers of latency. They are also involved in maintaining latency.
AST is expressed in latently infected cells and in productively infected cells.
AST recruits PCR2 to the 5' LTR and promotes LTR silencing.
Of note, AST is an HIV-encoded inducer of latency.
The future work will try to obtain miniaturized or chimeric AST-based non coding RNA molecules for permanent silencing of HIV.
Dr Symons (Melbourne) analyzed integration sites of HIV with a technique suitable for robotic. During the experiments new events were still observed despite the presence of raltegravir, arguing for ongoing residual viral replication.
Dr Beliakova-Bethell (La Jolla) compared the effects of SAHA to those of Romidepsin.
Romidepsin is a more profound and stable repressor of HDAC. It shows a greater up regulation of HIV transcriptional activators HSPA1A and KDM1A. It also down regulates repressors such as AES and ARID 13.
Finally, Dr Spina (San Diego) showed that latency is established preferentially in minimally activated and non dividing cells, i.e. just when the cells were infected and not later at a time when they are going back to a resting state.
SESSION 3: Virology of HIV persistence
John Coffin (Boston) spoke about provirus integration sites.
In treated patients, the virus that rebounds when therapy is stopped is the same as before therapy. However, different clonal populations of RNA and DNA appear after years on continuous therapy.
In long lived cells, sites of integration are determined both by initial preference ("hot spots") and by selection after integration.
He described a patients on ART, with 2 periods of treatment interruption, and final viral rebound despite ART. Integrations of proviral DNA were found in MKL2 and BACH2 sites. There was also integration in an ambiguous location, calles AMBI-1.
AMBI-1 provirus was intact and found responsible for the monomorphic clonal population found in plasma.
This patient died of cancer and had an autopsy.
Cells with AMBI-1 provirus were widely distributed and enriched in tumor tissue.
So, despite viral cytopathic effects and immune-mediated killing of expressing cells, cellular clones can both expand and harbor intact proviruses
Dr Howell (West Point) described the adaptation of the commercial assay Quanterix Simoa to measure p24 in plasma and cell lysates. It has a broad dynamic range and a limit of detection of 3 fg/ml.
She showed that HDAC inhibitors can increase p24 in ART suppressed patients CD4+ T cells ex vivo.
Using samples from David Margolis study using multiples doses of Vorinostat, she was able to show an increase in p24 plasma levels.
Dr Romerio (Baltimore) describe the adaptation of the Prime Flow RNA technology based on flow cytometry to measure RNA by using hybridization with several probes.
The limit of detection is about 10 RNA copies/cell and it can detect infected cells as low as 10-4 - 10-5.
Dr Hataye (Bethesda) showed modeling of HIV production (rate and duration; instantaneous bursts or sustained release) and concluded that sustained release accounts for >90% of HIV production. The release rate is 2400 HIV RNA copies/cell and the release duration of 3-4 days.
Dr Garcia Lerna (Atlanta) showed data on a simian model of PrEp using SHIV. For those animals who are infected despite PrEp, viremia is rapidly controlled, DNA levels are reduced compared to controls but this is transient and no longer found after 1 year, no difference in found in the size of the lymph node mononuclear cells reservoir.
Dr Kearney (Frederick) showed the presence of unspliced HIV RNA in expanded CD4+ T cell clones containing defective or replication competent proviruses.
5 patients on ART for 5-19 years were analyzedand at least 13% of expanded clones harbored unspliced HIV RNA. However, only a small fraction of cells (0.3%) contains replication competent viruses.
SESSION 4: Anatomic and non-CD4 cell reservoirs
Dr Farber (New York) presented the tissue localization of human T cell responses. She showed that TRM, effector memory cells found in tissues, are different from TEM found in blood.
She used a research consented organ donor database and showed that the distribution of T cells in tissues does not correlate with age but to tissue specificity.
Naive cells are found only in lymphoid tissues.
Cells that are in each tissue express different markers.
She then analyzed the distribution in patients sero+ for CMV and found 4 patterns of distribution:
-presence of cells in blood > others
-bone marrow or lung > others
-lymph nodes > others
-low frequency of these specific cells (<2%)
Dr Kim (Norfolk) showed a macaque model of lentiviral encephalitis and demonstrated proliferation (KI 67, CD163, incorporation of a thymidine analog) of perivascular macrophages.
Dr Gludish (Ithaca) conducted a study in Malawi with FISH:FACS assay and found replication competent HIV in the airways of ART suppressed patients. It is probable that alveolar macrophages are self maintained for a long time and low activation.
This shows that not all reservoirs need to be latent.
Dr Lamers (Los Angeles)showed that in a series of autopsies, HIV diversification and expansion was temporally related with metastasis. These viruses did not show resistance mutations.
Dr Routy (Montreal) presented the "Orchid study" collecting testis from 5 HIV-infected patients with gender reassignment and 10 controls.Oestrogens were stopped 4 week prior to surgery and HIV+ patients were undetectable in plasma viremia for at least 6 months.
This preliminary report concerns the fact that testis has immune privilege and contains immunosuppressive cells.
In both HIV+ and HIV - patient, Routy found:
-lower % of CD4 cells in testis/blood
-higher % CD8 cells in testis/blood
-increase in effector memory in testis/blood
-increase in expression of CCR5 in testis/blood
-increase status of immune activation in testis/blood
-increase of immunosuppressive CD39+ Tregs in testis/blood
-higher expression of IDO 1 and IDO 2 in testis/blood.
Data on the testis as a viral reservoir are ongoing.
Finally, Dr Hong (Pittsburgh) further emphazised that HIV persists in macrophages obtained from broncho-alveolar lavage of patients on suppressive ART harbour HIV. In 7 aviremic patients, no HIV RNA was found in these alveolar macrophages although HIV DNA was present in 6 out of 7 at levels close to those found in PBMC.
This cannot be explained by phagocytosis or CD4 contamination
Key words: HIV cure, HIV persistence, HIV reservoirs, HIV workshop