Research project P6/41 (Research action P6)
Therapy success has reduced acquired immunodeficiency syndrome (AIDS)-related morbidity and mortality significantly during the last years, but resistance to existing drugs is a growing problem. Novel strategies are needed, which should target the human immunodeficiency virus (HIV) in various ways and prevent treatment escape. To this end, fundamental knowledge needs to be acquired on more efficient use of chemical compounds, targeting “classical” viral enzymes, but also by looking at new targets, using molecular biological and immunological approaches.
We will investigate the possibility of inhibiting HIV replication using the following strategies:
1. Development of advanced bio-informatics tools to predict and counter viral escape under drug and immune pressure.
2. Finding new drug targets that do not suffer from cross resistance with current drugs.
3. Refining the RNA interference (RNAi) technology to prevent escape.
4. Exploring the possibilities of “autologous” immunotherapy, based on transfection of dendritic cells (DC) and other antigen-presenting cells (APC) with HIV-derived messenger RNA (mRNA).
5. Development of a suitable small animal model to test these new approaches in vivo.
In the present proposal, we will not evaluate new therapies in the clinic, but will perform the necessary fundamental research, using a multidisciplinary approach. Therefore, we propose a collaboration between different Belgian partners with a complementary competence, including a partner from the Antwerp University (Zwi Berneman, coordinator), the KULeuven (Anne-Mieke Vandamme), the University of Liège (Michel Moutschen) and the Ghent University (Bruno Verhasselt, a young investigator), a Belgian Federal scientific institution (Guido Vanham, Institute for Tropical Medicine) and a European research institution (Ben Berkhout, University of Amsterdam).
1. Developing bioinformatics tools to use the current drugs more wisely
Current guidelines recommend genotyping the drug-targeted viral protease and reverse transcriptase enzymes before making treatment decisions (Vandamme AM et al. Antivir Ther 2004;9:829-48). However, genotypic interpretation of drug resistance needs to be improved. Partner Vandamme is continuously updating the widely used Rega algorithm that her group developed (Van Laethem K et al. Antivir Ther 2002;7:123-9). Within this project, large datasets of patient treatment and resistance information (currently over 23000 patients), and virus evolutionary knowledge will be joined using datamining and modelling approaches to build a bioinformatics tool that can interpret for an individual patient the current resistance to drugs, but that can also predict for this patient the evolution of future resistance towards a next line drug regimen. In this way information will be developed on optimal sequencing of treatment combinations with minimal resistance cost, thus allowing a long-term treatment strategy. In addition, such bioinformatics tools will be able to map particular resistance pathways to be confirmed by in vitro classical molecular biological techniques (in vitro mutagenesis, genotyping, phenotyping). This approach is entirely new, but preliminary evaluations suggest that the concept is workable (Abecasis et al. AIDS 2005;19:1799-1806). This integrated fundamental research on viral evolution under drug pressure provides a model to explore similar, more elaborate bio-informatics tools to predict and counter viral escape under immune pressure. Any progress in this new area will be beneficial for future immunotherapeutic strategies (see partners Vanham and Berneman).
2. Nef as a new therapeutic target
Virus-cell interactions are interesting drug targets since less drug resistance is expected, as interaction sites of viral and cellular proteins are likely to be conserved. The HIV-1 Nef protein plays an important role in the disease development towards AIDS. In vitro, Nef does not seem to be essential in the virus replication cycle, however patients infected with Nef-deleted virus are progressing to AIDS much slower. Nef could therefore be an interesting target to slow down disease progression. Nef interferes with the differentiation of T-lymphocytes by interacting with domains important for T cell receptor signalling (Stove V et al. Blood 2003;102:2925-32). Recent findings show that CD4, major histocompatibility complex (MHC) class I and CD8 are internalised by Nef (Stove V et al. J Virol 2005;79:11422-33). Partner Verhasselt will investigate in depth the mechanisms, involved in the ability of Nef to disturb T cell development and to interfere with the function of both DC and T cells (with partners Berneman and Vanham). Dedicated RNAi technology will be developed with partner Berkhout. An additional emphasis will be on the relation between Nef sequences of primary isolates and their function (link with datamining techniques of partner Vandamme). This knowledge is the fundamental requirement to identify which interactions between Nef and cellular proteins should be targeted by antiviral therapy.
3. RNAi as a novel treatment strategy
Partner Berkhout recently showed that RNA interference (RNAi) is highly effective in suppressing HIV-1 replication in vitro (Berkhout B. Curr Opin Mol Ther 2004;6:141-5). He will develop a durable lentiviral gene therapy that should protect HIV-susceptible cells by expression of multiple short-interfering RNAs (siRNAs) against the viral RNA genome. Potent inhibition by some siRNAs has been shown, but virus escape through mutation of the target sequence or alternative pathways has been described (Das AT et al. J Virol 2004;78:2601-5). To prevent viral escape, partner Berkhout intends in this project to take a multi-siRNA approach similar to the combined use of antiviral drugs in highly active anti-retroviral therapy (HAART). With this gene therapy we aim to provide long lasting improvement of the condition of HIV-1-infected patients, especially those that cannot be treated anymore with antivirals due to viral resistance or drug toxicity. HIV-1-infected patients in The Netherlands are mainly native males infected with HIV-1 subtype B strains, but a fast growing group of immigrant females and males with HIV-1 subtype C are diagnosed. Worldwide, females and males having HIV-1 subtype C constitute the fastest growing group. All HIV-1 subtypes are potentially targeted by our future gene therapy approach. In this pre-clinical project, we will test the potency of different RNAi approaches and viral escape possibilities. Partner Berkhout will also try to silence cellular factors that are critical for virus replication, or that are demonstrated to interact with Nef by the research of partner Verhasselt. In addition, in collaboration with partners Vanham and Berneman, RNAi will also be applied to inhibit the “suppressor of cytokine signaling” (SOCS) pathway in an attempt to improve HIV antigen presentation.
4. Immunotherapy as a novel treatment strategy
Immunotherapy is meant to boost the patient’s own immune reaction against HIV and prevent viral replication, evolution and escape. Partner Vanham together with partner Berneman are currently establishing the basis for a new therapeutic fully autologous vaccination with monocyte-derived DC (MO-DC) and HIV protein-encoding mRNA. They recently showed that both plasma viral and cellular proviral autologous HIV sequences from individual patients could be amplified, transcribed into mRNA, and used to load MO-DC in order to specifically trigger autologous CD4+ and CD8+ T cells (Van Gulck ER et al. Blood 2006;107:1818-27). The proposed project will involve loading DC with proviral-derived mRNA from HIV-1-infected patients in comparison with consensus sequences. Their capacity to induce T cell responses with protective potential will be assessed, using a combination of in vitro methods. Secondary responses will be studied in DC/T cell cultures from patients with low to undetectable viral load, either long term non-progressors (LTNP) or under HAART. For optimization of conditions, expert knowledge on how DC can be used and modified for this purpose, partner Berneman developed techniques to isolate, manipulate and transfect DC (Van Tendeloo VFI et al. Blood 2001;98:49-56), including in the HIV field (Van Gulck ER et al. Blood 2006;107:1818-27). Building on the findings that were made on MO-DC classically grown in granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin (IL)-4-containing medium, partner Berneman (in close collaboration with partner Vanham) will investigate whether modification of the DC differentiation- and/or maturation-inducing factors and addition of structural as well as regulatory HIV sequences (as antigens) will result in improved HIV-1-specific T cell responses ex vivo. In addition, other APC systems (e.g. activated B lymphocytes) will be explored to efficiently boost T-cell responses ex vivo. The aim will be to obtain efficient CD4+ and CD8+ memory responses against HIV (Van den Bosch GA et al. J Immunother 2006;29:107-21), which should prevent viral rebound and escape. Finally, we hope that optimization of the APC function will allow to generate primary in vitro responses (in peripheral blood lymphocytes (PBL) from seronegative subjects), thus contributing to progress in the prophylactic vaccination field as well.
5. An in vivo small animal model
Within the project we will move from in vitro cell culture infections towards novel in vivo mouse model systems. Partner Moutschen is developing a Trimera mouse model, which allows a better engraftment and a longer survival of human PBL than the classical NOD/SCID mice. Trimera mice are more convenient for reconstitution with PBL from infected patients and for subsequent (immune) therapeutic studies. In a first step, the kinetics of HIV replication will be studied after complementation of the mice with PBL from long-term non-progressors (LTNP) and HAART patients. Next, we will evaluate the in vivo antiviral activity of adoptively transferred T cell lines, generated in vitro from these patients by partner Vanham. In addition, the prophylactic efficiency of APC-based vaccines (developed by partner Berneman) will be evaluated in Trimera mice reconstituted with normal PBL and challenged with HIV after vaccination. Finally, the therapeutic efficiency of APC-based vaccines in mice reconstituted with PBL from HIV-infected donors will also be studied. Clearly, these experiments will provide the proof-of-concept for a more extended use of this model in therapeutic trials with novel agents, including RNAi (partner Berkhout).
