Cure Strategies: "Viral and Immune-Targeted Interventions: Hit Me with Your Best Shot" - Oral Session at IAS 2013 Kulala Lumpur June 30-July 3
Reported by Jules Levin
from Jules: there were 4 oral slide presentations in this session on cure strategies.
Destruction of the residual active HIV-1 reservoir by Env-specific immunotoxin
Presented by J. Victor Garcia (United States).
P.W. Denton1, J. Long1, N. Archin1, S. Wietgrefe2, S. Choudahry1, C. Sykes3, K. Yang3, M.G. Hudgens4, I. Pastan5, E.A. Berger6, A. Haase2, A. Kashuba3, D.M. Margolis1, J.V. Garcia1
1University of North Carolina at Chapel Hill, Internal Medicine/Infectious Diseases, Chapel Hill, United States, 2University of Minnesota, Minneapolis, United States, 3University of North Carolina at Chapel Hill, School of Pharmacy, Chapel Hill, United States, 4University of North Carolina at Chapel Hill, Gillings School of Global Public Health, Chapel Hill, United States, 5National Cancer Institute/NIH, Bethesda, United States, 6National Institute of Allergy and Infectious Diseases/NIH, Bethesda, United States
Written by J. Victor Garcia for NATAP:
Last year we published a study that validated BLT humanized mice (Bone Marrow/Thymus/Liver) for the study of HIV latency (Denton et al JVI 2012), a mouse with a human hematopoietic/immune system that is susceptible to HIV infection and responds to ART. Here we build on this first to establish a good ART regimen that would show good penetration in tissues. We found that a combination of Tenofovir, emtricitabine and Raltegravir was able to suppress viremia in plasma of infected BLT mice to undetectable levels and in addition, that it penetrated well into all the tissues we examine.
Secondly, we then asked whether or not there was any residual HIV RNA expression in those tissues. The results show that despite the strong drug penetration there was residual viral RNA in all tissues examined. This is what has been called the "residual active reservoir". Since these HIV producing cells could contribute to both viral rebound and the re-seeding of the long lasting latent reservoir there is a great deal of interest in their elimination.
Here is where the immunotoxing comes into play. Immunotoxins are antibodies that bind a cell surface receptor on cells. However, unlike normal antibodies, immunotoxins have a "payload" that after entering the cell is released. This released payload is lethal to the cells where it enters and is released. Immunotoxins have been extensively used in cancer treatment and one of the best know is in Ontak that is used to treat cutaneous T cell lymphoma.
So in the third part, we used an immunotoxin that combines an antibody that binds the envelop protein of HIV expressed on the cell surface of infected cells with a toxin molecule from pseudomomonas. The key experiment was to add the immunotoxin to the ART regimen and to see if that would reduce the active reservoir. The answer is yes. We saw significant reductions in the levels of both viral RNA and HIV+ cells after this combination treatment in all tissues examined.
The implications of these result go beyond the fact that this approached was able to reduce the active reservoir. Specifically, virtually all approaches to eradicate HIV involve two steps: induction of the virus from latency (also referred to as kick) and destruction of the infected cell (or kill). Originally, most people thought that simply inducing HIV would do the trick (kill). Recent reports suggest that is not the case and that the HIV expressing cells might not die on their own. Therefore, a killing strategy is needed.
So the good news is that immunotoxins would work at the kill part. However, what is even more important is that what we have established is a "platform" where virtually any new approach to kill HIV infected cells can be evaluated and directly compared with any other approach. It can be a cellular based approach, an immunology based approach or a drug based approach. It does not matter. In addition, it can be combined with an induction treatment like recently done for vorinostat. Therefore this new platform will help inform future clinical trials what strategies might be best for testing in patients.
Background: HIV reservoirs are responsible for HIV persistence in patients during antiretroviral therapy (ART). Persistence of HIV infection despite ART is marked by two phenomena - the persistence of quiescent but replication-competent provirus and the persistent production of HIV RNAs by an undefined population(s) of cells. This work demonstrates the efficacy of a novel targeted cell killing approach to deplete productively infected cells in vivo.
Methods: Humanized BLT mice were constructed, HIV infected, and treated with ART essentially as we have previously described (Denton et al. J. Virol 2012) or not treated with ART. Pharmacokinetic analysis and determinations of latently infected cells were performed as previously described (Choudhary et al. J. Virol 2010, 2012). An immunotoxin targeting HIV Env (3B3-PE38)(Bera et al. Mol Med, 1998) or vehicle was administered every other day for 14 days to ART treated animals. Animals were harvested and levels of residual RNA in tissues were compared between control, ART treated and ART + 3B3-PE38 treated mice using a combined mixed effect statistical model. In addition, individual infected cells were quantified using in situ hybridization and compared between experimental arms using the Mann-Whitney test.
Results: We examined HIV reservoirs in BLT humanized mice during ART and performed a tissue-specific pharmacodynamic analysis that reveals the effect of antiretrovirals on systemic HIV-1 RNA levels at the individual tissue level. Despite strong systemic HIV suppression in plasma (p< 0.001), HIV expression in tissues continued to persist. To eliminate HIV producing cells, we augmented ART with an immunotoxin targeting HIV Env (3B3-PE38) and found that addition of the immunotoxin further reduced RNA levels up to 3.2 logs in individual tissues (p< 0.001). This result was confirmed by in situ hybridization. This significant reduction in cell-associated HIV RNA production over ART alone highlights the susceptibility of the residual active HIV reservoir to targeted cytotoxic therapy.
Conclusion: Our in vivo data show that complementing ART with an immunotoxin targeting HIV-producing cells leads to a dramatic further reduction in the levels of productively infected cells in all tissues analyzed. These results suggest an effective strategy to eradicate HIV with a combinatorial approach.
Targeting HIV-1 persistence in CD4 T memory stem cells by pharmaceutical inhibition of beta-catenin
M. Buzon1, H. Sun1,2, C. Li1, E. Martin-Gayo1, A. Shaw1, E. Rosenberg3, F. Pereyra1, B. Walker1, X. Yu1, M. Lichterfeld3
1Ragon Institute of MGH, MIT and Harvard, Cambridge, United States, 2AIDS Research Center, China Medical University, Shenyang, China, 3Massachusetts General Hospital, Department of Medicine, Boston, United States
Written for NATAP by Mathias Lichetfeld
An important aspect of HIV-1 infection is that the virus can persist for extremly long time in the human body. Even when active viral replication is suppressed for more than 10 years, rebound of HIV-1 occurs rapdily once treatment is discontinued. Where and how HIV-1 can persists for such long periods of time is an unresolved question- after all, there are not so many cell types that persist for that much time. For many years, it's been hypothesized that HIV-1 can persist in stem cells - cells that persist almost indefinitely in the human body, and have critical function for regenerating differentiated tissue cells. Hematopoietic stem cells have for a long time been suspected to serve as such a durable reservoir for HIV, but a number of studies have shown that in many cases, these cells do not harbor HIV-1. Recently, a new population of stem cells was discovered - a group of T lymphocytes termed "T memory stem cells". These cells represent the earliest and most immature population of memory T cells, and differ from all other T cells by an increased ability to proliferate and persist long-term. Emerging data suggest that HIV-1 can effectively infect these cells, and exploit their stem cell charactersitics for promoting long-term viral persistence. In fact, data presented at CROI 2013 and the IAS meeting in 2013 suggest that T memory stem cells from HAART-treated patients harbor high levels of HIV-1, and that the decline of HIV-1 DNA in these cells is extremly slow. This may imply that these cells represent one of the most durable and stable CD4 T cell populations that is likely to be an important barrier against efforts to eradicate HIV-1. One strategy to specifically target these cells would be to pharmaceutically inhibit molecular mechanisms that promote survival and long-term persistence of T memory stem cells. Similar approaches are currently being used in cancer patients, where drugs that specifically inhibit persistence of cancer stem cells are being investigated in early stage clinical trials.
Background:Treatment with antiretroviral combination therapy can effectively suppress active HIV-1 replication, but HIV-1 persists in the human body and rapidly rebounds after discontinuation of therapy. T memory stem cells (Tscm) represent a recently-discovered subpopulation of T cells that persist for extremely long periods of time and are maintained by a stem cell-like developmental program governed through the wnt/beta-catenin pathway. The role of CD4 Tscm for viral persistence is unclear.
Methods: PCR assays were used to determine the amount of cell-associated HIV-1 DNA in sorted CD4 T cell subsets from HAART-treated HIV-1 patients. The presence of replication competent virus within the CD4 T cell subsets was tested using viral reactivation assays. Phylogenetic association studies were performed with viral Env sequences amplified form plasma and individuals CD4 T cell subsets. Effects of the pharmaceutical beta-catenin inhibitor C-82, (the active metabolite of PRI-724, currently tested in clinical trials, e. g. NCT01606579) on CD4 T cell differentiation were studied using ex-vivo culture assays.
Results: HIV-1 DNA in CD4 Tscm from HAART-treated patients were high and exceeded HIV-1 DNA levels in alternative cell subsets. Viral reactivation assays demonstrated that CD4 Tscm harbor replication-competent virus. Viral sequencing studies revealed close phylogenetic associations between circulating plasma HIV-1 strains during early disease stages, and HIV-1 DNA isolated from CD4 Tscm after 6-12 years of therapy, consistent with long-term viral persistence in CD4 Tscm. In vitro culture assays demonstrated that pharmaceutical beta-catenin inhibitors can promote differentiation of CD4 Tscm into more short-lived effector CD4 T cells.
Conclusion: Tscm serve as a long-lasting reservoir for HIV-1 that importantly contributes to viral persistence. Targeting this specific cell compartment by pharmaceutical beta-catenin inhibitors may have an adjunct or additive role for reducing long-term viral persistence in CD4 Tscm.
DC infected by the ANRS MVAHIV vaccine candidate primes NK cells with anti-HIV specific activity through a mechanism involving NKG2D and NKp46 on NK cells, and membrane-bound IL-15 on DC.
Written for NATAP by Uriel Moreno-Nieves
(ppt slides attached)
U.Y. Moreno Nieves1, J.-S. Cummings1, V. Arnold1, A. Gilbert1, K. Yarbrough1, C. Didier1, Y. Levy2, F. Barre-Sinoussi1, D. Scott-Algara1, ANRS HIV Vaccine Network (AHVN)
1Institut Pasteur, Unite de Regulation des Infections Retrovirales, Paris, France, 2INSERM U955, Groupe Henri-Mondor Albert-Chenevier, Creteil, France
Authors: Uriel Y. Moreno-Nieves, Jean-Saville Cummings, Vincent Arnold, Adrien Gilbert, Kevin Yarbrough, Celine Didier, Yves Levy, Francoise Barre-Sinoussi, Daniel Scott-Algara and ANRS HIV Vaccine Network (AHVN).
Background: Natural Killer (NK) cells are the major antiviral cell population of the innate immune system. It is assumed that the innate immune response is non-specific by opposition to the adaptive immune response; however increasing evidence points out to the fact that NK cells can have some features of the adaptive immune system. The activity of NK cell can be modulated by the interaction with dendritic cells (DC). Using the HIV vaccine candidate MVAHIV, developed by the French National Agency for Research on AIDS (ANRS), we analysed whether or not stimulation of NK cells by MVAHIV-infected DC is able to induce an anti-HIV specific NK cell activity.
Results: We found that NK cells stimulated by MVAHIV-infected DC have a significantly better capacity to control HIV replication in autologous DC and CD4+ T cells when compared to NK cells stimulated by MVAWT-infected DC. The specificity of the anti-HIV activity was determined by measuring the NK cell activity against other virally infected cells; and also we analysed their ability to respond to tumor cell lines. In depth analysis of the mechanism underlying the specific stimulation of NK cells by MVAHIV-infected DC, showed that the engagement of two NK cell receptors, namely NKG2D and NKp46, modulates the subsequent anti-HIV NK cell activity. Also, we observed that membrane-bound IL-15 (mbIL-15) and/or IL-15 are necessary for the induction of the specific anti-HIV NK cell activity.
These data demonstrate that the MVAHIV vaccine candidate is able to induce a specific anti-HIV-1 activity of NK cell following their interaction with MVAHIV-infected DC, and that the acquisition of such antiviral activity relies on a modulated crosstalk involving NKG2D and NKp46 on NK cells and the regulation of mbIL-15 on infected DC. This data joins the mounting evidence showing that NK cells can have some features of the adaptive immune system, such as a targeted specific response.
Future experiments: We plan to further analyse the mechanism by which NK cells can acquire an anti-HIV specific activity after stimulation by MVAHIV-infected DC.
The HIV-1 splicing inhibitor, SPL-464, compromises viral replication in vitro and induces a long lasting anti-viral effect in humanized mice infected with HIV-1
ppt oral slide presentation attached
Presented by Jamal Tazi (France).
Link to webcast: http://pag.ias2013.org/flash.aspx?pid=301 and
N. Campos1, R. Myburgh2, A. Garcel1, A. Vautrin3, D. Scherrer1, M.A. Wainberg4, R. Speck2, J. Tazi3
1Splicos, Montpellier, France, 2University Hospital of Zurich, Zurich, Switzerland, 3University of Montpellier-IUF, IGMM-CNRS UMR 5535, Montpellier, France, 4McGill University, Montreal, Canada
Successful production of new infectious viruses from proviral genomes integrated into human blood cells requires an adaptation of the viral splicing machinery to allow expression of both spliced and unspliced viral mRNAs. Therefore, targeting HIV-dependent alternative splicing represents an approach to potentially target viral reservoirs as well as to treat HIV infection.
In collaboration with SPLICOS Inc., we designed, synthesized and screened 800 analogs of a polycyclic indole compound (IDC16) previously shown to interfere with the exonic splicing enhancer activity of the splicing factor SRSF1 and to compromise the assembly of infectious particles (bakkour et al 2007). Five compounds were selected, among which SPL-464 demonstrated unique and very promising properties. In brief, SPL-464 inhibited HIV replication in macrophages and (PBMCs from several donors), replication of HIV-1 and HIV-2 of varying T cell-tropism, as well as clinical isolates of different subtypes (B and C) at the nanomolar concentrations. SPL-464 did not select for drug resistance and deep sequencing demonstrated an absence of any possible mutations that might have emerged under SPL-464 treatment. In vitro, SPL-464 protected CD4+ cell counts in infected cultures and allowed an increased CD4+/CD8+ ratio.
Furthermore, SPL-464 had no adverse effect on global pre-mRNA splicing in PBMCs, while it specifically modulated HIV splicing. This selectivity was also demonstrated by the ability of SPL-464 to modulate the expression of a single miroRNA that can be used as a biomarker of efficacy in vivo.
Proof of concept of SPL-464 n vivo,efficacy was demonstrated in two animal models of retroviral pathogenesis using replication-competent HIV-1. In these models, SPL-464 alone, administered by gavage, efficiently compromised viral multiplication, whereas a combination of 3TC, Raltegravir and Tenofovir (ART) was required to achieve viral inhibition. Although a rebound of viral load was observed in humanized mice treated with ART after one week of treatment interruption, only a slight or no viral rebound was observed in mice treated with SPL-464 at two months following treatment interruption , consistent with a potential effect on viral reservoirs.
SPL-464 is an anti-HIV-1 molecule that can interfere with proviral cDNA transactivation and may challenge the vitality of HIV reservoirs.
Track A report by Nichole Klatt
Viral and Immune-Targeted Interventions: Hit Me with Your Best Shot
Victor Garcia: Destruction of the residual active HIV-1 reservoir by Env-specific immunotoxin
Experimental Goal: To eliminate cellular reservoirs of HIV that continue to actively produce virus in tissue compartments despite suppression of plasma viremia with ARVs
-Assessed tissue penetration of ARVs in vivo in BLT mice
-3B3-PE38 immunotoxin (antibody directed to env and carrying pseudomonas toxin) to reduce HIV RNA
-ARVs + 3B3-PE38 reduced HIV RNA as much as 1,000 fold in tissues and overall 0.8 logs
-mechanism is loss of productively infected cells
Mathias Lichterfield: Targeting HIV-1 persistence in CD4 T memory stem cells by pharmaceutical inhibition of beta-catenin
-HIV is persistent in CD4+ T stem cell memory cells (Tscm) and central memory (Tcm) cells
-Hierarchy of genetic distance in different CD4+ T cells that demonstrates Tscm may be longest lived and most durable reservoir
-HDACi turns HIV genes on
-Beta catenin inhibitors induce differentiation of Tscm and Tcm
-synergize HDACi and Beta-catenin inhibitors to eradicate HIV?
Uriel Moreno-Nieves: DC infected by the ANRS MVAHIV vaccine candidate primes NK cells with anti-HIV specific activity through a mechanism involving NKG2D and NKp46 on NK cells and membrane-bound IL-15 on DC
-NK cells stimulated by MVAHIV - infected dendritic cells (DC) are able to control virus infection of DCs
-Can NK cells control infection in other cell types?
-MVA priming increases control of HIV infection by NK cells
-Modulated by NKG2D
-Blockade of NKG2D decreases membrane bound IL-15
-IL-15 is important for MVA priming of NKs
-Suggests NK cells may be important in vaccine efficacy
Jamal Tazi: The HIV-1 splicing inhibitor, SPL-464, compromises viral replication in vitro and induces a long lasting anti-viral effect in humanized mice infected with HIV-1
-Alternate splicing of HIV after cell infection initiates replication
-HIV-1 RNA has weak 3' splice sites, dependent on regulators that target HIV sequences
-Splicos has a library of small chemical compounds targeting splicing machinery, including SPL-464
-SPL-464 inhibits virus replication in macrophages from human donors
-SPL-464 reduces viral load & rescues CD8/CD4 ratio in humanized mice