Novel drugs and treatment strategies for HIV-1

Abstract

XIX International Conference of the International AIDS Society

Washington DC, USA, 22–27 July 2012

Exploring new approaches in the development of HIV-1 therapeutics is an important component in a multifaceted approach to combating global HIV-1/AIDS-related mortality. This meeting report describes novel concepts of three eminent investigators speaking at a session of the XIX International Conference of the International AIDS Society. This conference attracted approximately 24,000 delegates comprising patients, scientists, clinicians and other key stakeholders. Herein, three putative targets of novel therapy are described: initial CD8-positive T-cell responses to HIV-1 infection, integration of HIV-1 provirus and removal of proviral DNA from host cells.

Keywords::

• AIDS
• CD4
• CD8
• hematopoietic
• HIV-1
• integrase
• LEDGF
• LEDGIN
• T-cell receptor
• tre-recombinase

While rollout of conventional antiretroviral therapy to areas of high prevalence has helped to reduce AIDS-related morbidity and mortality [101], researchers continue to develop novel strategies in the hope of contributing towards eventual functional, if not sterilizing, cure. It was noted throughout the scientific sessions that while global eradication of HIV-1 cannot come from therapeutic measures alone, such strategies must play a key role in reducing the prevalence of HIV-1.

Concepts of ‘treatment as prevention’, and treatment in concert with nonpharmacologic prevention modalities, which were explored throughout the conference, further justify research into new pathways and novel antiretrovirals. The concepts of three investigators pertinent to the development of HIV-1 therapeutics, at the ‘Novel Drugs and Treatment Strategies’ session (24 July 2012) of the XIX International AIDS Society Conference (Washington, DC, USA, 22–27 July 2012), are summarized in this article [102].

Enhancing initial CD8-positive T-cell responses

Scott Kitchen (David Geffen School of Medicine at University of California, Los Angeles, CA, USA) reminded us that HIV-1 infection generates a very potent T-cell response, with incomplete dysfunctional killing of infected cells by CD8-positive T cells being a hotly debated issue. His group has developed molecular cloned HIV-1-specific T-cell receptors (TCRs) to modify hematopoietic stem cells (HSCs), aiming to explore the relationship between virally infected cells and CD8-positive T cells. They have cloned 30 HLA Class I-restricted molecular clones – that is, TCRs specific to HIV-1 components such as Gag, Vpr and others in the context of HLA-restricted responses [1].

Kitchen et al. discussed a proof-of-principle study using a gag sl9 epitope Class I HLAB201*-restricted TCR clone. A class A201* restricted TCR with unknown antigen specificity was used for control. In a modified humanized mouse model, fetal liver-derived CD34 HSC progenitors modified with a lentiviral vector containing the anti-HIV-1 TCRs or control TCRs were used. These cells were combined with fetal thymus tissue and liver stromal tissue and implanted under the kidney capsule. Following a successful two-stage engraftment process, mice were injected with HIV-1, which was modified to contain a reporter gene to confirm successful infection [1].

Kitchen’s group observed transgenic TCR expression in the bone marrow, thymus, liver, spleen and peripheral blood throughout infection. At 6 weeks, significant reductions of HIV-1-expressing cells were observed in mice containing the transgenic HIV-1-specific TCRs compared with control TCR mice. A significant preservation of CD4-positive T cells in mice containing the anti-HIV-1 TCRs versus control mice was noted. Ultrasensitive quantitative PCR methods showed significant viral RNA reductions in the anti-HIV-1 TCR group compared with the control group at weeks 2 and 6 [1,2].

Kitchen et al. described little genetic difference between input HIV-1 and sampled HIV-1 throughout infection in both groups. Curiously, this suggests that immunologic pressure from the anti-HIV-1 TCRs did not drive viral evolution. At week 6, Kitchen et al. found significant reductions in viral RNA in the spleen, bone marrow and thymus of the anti-HIV-1 TCR group. Flow cytometric gating for specific T-cell populations demonstrated effector memory differentiation in anti-HIV-1 TCR mice compared with the control, which displayed a predominant naive T-cell phenotype. In addition, mice with the highest level of antigen-specific precursors had lower levels of viral RNA. The ultimate aim of Kitchen et al. is to develop a bank of HIV-1-specific TCRs that can be used as part of a therapeutic strategy in early HIV-1 infection [1,2].

Blocking HIV-1 integrase

LEDGF/p75 was first described as a cofactor of HIV-1 integrase, which tethers provirus to the cell genome. Frauke Christ (Katholieke Universiteit Leuven, Leuven, Belgium) explained how LEDGF/p75 has a well-defined interaction domain with HIV-1 integrase [3]. The team developed the ‘LEDGINs’ – that is, LEDGF/p75: the first in their novel class of antiretrovirals that would bind to the integrase binding site for LEDGF/p75. Using the pharmacophore model, combining medicinal chemistry and structural biology, the group developed different series of these compounds to block the LEDGF/p75-integrase binding site, associating with an allosteric site on HIV-1 integrase [4].

Christ’s group have recently developed an additional series of LEDGIN compounds with higher potency, such as cx14442, which displays nanomolar activity in cell culture assays with very high selectivity. Her group focused on exploring multiple mechanisms of LEDGIN action. Christ’s group performed a standard transfer assay, in which LEDGIN was added to integrase preassembled on HIV-1 DNA, observing partial integrase inhibition. After reversing the experimental design, using integrase prebound to LEDGIN followed by long terminal repeat (LTR) sequence of an HIV-1 oligonucleotide, integrase inhibition increased five- to six-fold [5].

Christ et al. used the differential scanning fluorimetry thermostability assay to investigate melting curves of integrase showing very strong effects (inducing an increase of 14°C) with their most potent LEDGIN candidate compounds. This supported the concept that LEDGINs may strongly stabilize the oligomeric state of integrase. Further experiments demonstrated that a 12-h delay in LEDGIN addition leads to 50% loss of integrase activity, supporting the activity of LEDGIN at the integration step of HIV-1 replication [5].

Christ’s group explored whether LEDGINs display cross-resistance with conventional integrase inhibitors, testing against a broad spectrum of raltegravir-resistant HIV-1 subtypes, observing that LEDGINs potently inhibit replication of these subtypes. Compared with older LEDGIN candidates, using newly developed and more potent LEDGIN compounds prevented an integrase single-point mutation from overcoming LEDGIN-induced inhibition. Christ et al. then performed combination studies showing an additive effect of LEDGIN and raltegravir in cell culture, verging on synergistic effects [5].

Christ’s group produced viral particles from persistently infected cells in the presence of LEDGINs, raltegravir or control (dimethyl sulfoxide). The cells could produce mature viral particles in the conditions of dimethyl sulfoxide, raltegravir and the LEDGIN boost. However, when the viral particles produced in the presence of LEDGIN and raltegravir were used to infect HIV-1-naive cells, LEDGINs significantly reduced infectivity [5].

Christ summarized LEDGINs as a new class of antiviral drugs that have a multimodel mechanism of action on HIV-1 integrase. They accepted that more data are required to confirm a direct microbicidal effect of the LEDGIN compounds, and it was suggested that dilutional studies may be of use in exploring this specific mechanism of LEDGIN action as a disruptor of viral particles. Nevertheless, this new class of HIV-1 integrase inhibitors shows great promise for future study.

Excising HIV-1 provirus

When HIV-1 proviral DNA has integrated so far, there is no therapeutic agent that can actively remove it. Helga Hofmann-Sieber’s group (Heinrich Pette Institute – Leibniz Institute for Experimental Virology, Hamburg, Germany), in collaboration with researchers from across Europe have generated an HIV-1 LTR sequence-specific recombinase termed tre-recombinase that is able to the excise proviral genome from an infected cell. Tre-recombinase is derived from the well-established Cre–Lox system and recognizes a 34-bp asymmetric sequence of the LTR region specific for a natural HIV-1 isolate. Hofmann-Sieber and colleagues explained that this tre-recombinase removes provirus from HIV-1-infected cell culture. More potent forms of delivery for tre-recombinase were explored, and Hofmann-Sieber and collaborators eventually designed an advanced lentiviral self-inactivating vector with a very good safety profile [6].

This vector expresses green fluorescent protein as a reporter gene, and a tre-recombinase under an HIV-1-derived promoter. Hofmann-Sieber described how tre-recombinase is only expressed in the presence of HIV-1 Tat promoter. Hofmann-Sieber confirmed that tre-recombinase expression is not cytopathic or genotoxic, through investigating long-term toxicity in Jurkat T cells, reactivation potential of primary CD4-positive T cells, whether the differential potential of human HSCs would be affected by the presence of tre-recombinase, as well as in vivo toxicity analysis in the murine model [6].

Using humanized mice repopulated with human primary CD4-positive T cells and human HSCs, Hofmann-Sieber and colleagues explored the effect of tre-recombinase on HIV-1 infection. In the primary T-cell arm, CD4-positive T cells were isolated from peripheral blood, transduced with lentiviral vector or control, transplanted into humanized mice and then checked for engraftment 8–20 weeks later. The mice were then infected with HIV-1 and the progress was monitored for 16 weeks. In the tre-recombinase group, viral load reduced significantly, while for the control group, the viral load was stable or somewhat increased [6].

In the tre-recombinase group, human T cells were seen to be significantly increasing in population, while the T-cell population in the control group remained stable. Histologically, Hofmann-Sieber and colleagues observed high T-cell concentrations in the spleen, finding significantly lower levels of HIV-1 p24 core antigen compared with the control group. They then isolated HSCs from human cord blood, and transduced these with lentiviral vectors using similar methods to that of the CD4-positive T-cell arm. Once again, for tre-recombinase-treated mice, p24 levels were close to zero, while a significant level of p24 was observed in the control group with similar histological observations [6]. While Hofmann-Sieber and colleagues conceded that transduction efficiency for CD4-positive T cells and HSCs was 80 and 50%, respectively, this research is highly promising. Hofmann-Sieber and colleagues suggested that tre-recombinase gene therapy may become coupled to a chemotherapeutic purging approach, which cannot in isolation lead to eradication of the virus.

Conclusion

This report has described three strategies acting at intensively researched stages in the HIV-1 lifecycle. The focus of HIV-1 immunotherapy research is moving towards targeting the initial events of HIV-1 establishment, such as proviral integration, leading to viral persistence or the early dysfunctional CD8-positive T-cell response. Early excision of proviral HIV-1 DNA, once integration has occurred in a small number of target cells, could reflect a new stratagem in postexposure prophylaxis. Instead of modifying the immunologic environment following disseminated infection and establishment, novel strategies that arm target or responding effector cells against HIV-1 infection prior to encountering the virus particle may represent a paradigm shift in efforts to combat the epidemic.

Acknowledgements

This conference was organized by the International AIDS Society.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.