HIV-derived vectors for therapy and vaccination against HIV.

Despite being at the origin of one of the world's most devastating public health concerns, the Human Immunodeficiency Virus (HIV) has properties that can be harnessed for therapeutic purposes. Indeed, the ability of HIV to efficiently deliver its genome into the nuclear compartment makes it an ideal vector for gene delivery into target cells. The design of so-called HIV-derived vectors, or more generally lentiviral vectors (LVs), consists in keeping only the parts of the virus that ensure efficient nuclear delivery while entirely removing all coding sequences that contribute towards the replication and pathogenesis of the virus: as a result, the vector genome is composed of less than 10% of the original virus genome and exclusively cis-active sequences. Proteins required for the formation of the lentivector particles and for the early steps of viral replication (including Gag- and Pol-derived proteins) are provided in trans. HIV-derived vectors are thus non-replicative virus shells that deliver genes of interest into target cells with high efficiency. Undoubtedly, there is a hopeful twist of fate in our fight against AIDS, which consists in using these vectors to achieve gene therapy and vaccination against HIV itself. This review summarises the current generation of LVs with a special focus on vaccine applications against AIDS. Preclinical data are very encouraging and efforts are ongoing to optimise these vectors, to increase their safety and improve their immunogenicity.

[Retroviral restriction factors : from Fv1 to TRIM5α]. / Les facteurs de restriction rétrovirale : de Fv1 à TRIM5α.

During their evolution, mammals have developed cellular factors interfering with retroviral replication, known as « restriction factors ¼. The prototype of these factors, Fv1, was characterized in the late 60's and blocks MLV infection. Some Fv1-like factors interfering with complex retroviruses, including HIV, have recently been discovered in primate cells. These restriction factors are referred to as Ref1, which blocksMLVreplication in human cells, and Lv1, which blocks the infection of non-human primate cells by various retroviruses, including MLV and HIV. These factors are all saturable by an excess of virus, target the viral capsid and interfere with an early step of viral replication. Lv1 and Ref1 have recently been found to be species-specific variants of a single protein called TRIM5α, a member of the TRIM protein family. The mechanism of action of these factors is still unknown. The existence of natural inhibitors of retroviral infection raises new hopes for the development of therapeutic tools against HIV infection.

Inhibition of HIV infection by the cytokine midkine.

The growth factor midkine (MK) has been reported to bind heparan sulfate and nucleolin, two components of the cell surface implicated in the attachment of HIV-1 particles. Here we show that synthetic and recombinant preparations of MK inhibit in a dose-dependent manner infection of cells by T-lymphocyte- and macrophage-tropic HIV-1 isolates. The binding of labeled MK to cells is prevented by excess unlabeled MK or by the anti-HIV pseudopeptide HB-19 that blocks HIV entry by forming a stable complex with the cell-surface-expressed nucleolin. MK mRNA is systematically expressed in adult peripheral blood lymphocytes from healthy donors, while its expression becomes markedly but transiently increased upon in vitro treatment of lymphocytes with IL-2 or IFN-gamma and activation of T-lymphocytes by PHA or antibodies specific to CD3/CD28. In MK-producing lymphocytes, MK is detectable at the cell surface where it colocalizes with the surface-expressed nucleolin. Finally, by using MK-producing CD4(+) and CD4(-) cell clones we show that HIV infection in cell cultures could be inhibited in both an autocrine and a paracrine manner. The potent and distinct anti-HIV action of MK along with its enhanced expression in lymphocytes by various physiological stimuli suggests that MK is a cytokine that could be implicated in HIV-induced pathogenesis.

The cell-surface-expressed nucleolin is associated with the actin cytoskeleton.

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface. Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect. Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum-Golgi complex. The cell-surface-expressed nucleolin becomes clustered at the external side of the plasma membrane when cross-linked by the nucleolin-specific monoclonal antibody mAb D3. This clustering, occurring at 20 degrees C and in a well-organized pattern, is dependent on the existence of an intact actin cytoskeleton. At 37 degrees C, mAb D3 becomes internalized, thus illustrating that surface nucleolin can mediate intracellular import of specific ligands. Our results point out that nucleolin should also be considered a component of the cell surface where it could be functional as a cell surface receptor for various ligands reported before.

The HB-19 pseudopeptide 5[Kpsi(CH2N)PR]-TASP inhibits attachment of T lymophocyte- and macrophage-tropic HIV to permissive cells.

The HB-19 pseudopeptide 5[Kpsi(CH2N)PR]-TASP[psi(CH2N) indicating a reduced peptide bond], which binds the cell surface-expressed nucleolin, is a potent inhibitor of HIV infection. Here, by using primary T lymphocyte cultures and an experimental cell model to monitor HIV entry, we show that HB-19 inhibits in a dose-dependent manner both T lymphocyte- and macrophage-tropic HIV isolates. Similar positively charged control pseudopeptides have no effect on HIV infection even at high concentrations. These observations, and the fact that HB-19 has no effect on SIV-mac and HIV-1 pseudotyped with VSV envelope glycoproteins, confirm the specific nature of this inhibitor against the entry process mediated by the HIV envelope glycoproteins. Finally, association of low doses of HB-19 with beta-chemokines or AZT results in an increased inhibitory effect on HIV infection. HB-19 has no inhibitory effect when added to cells a few hours after HIV entry. On the other hand, in HB-19-pretreated cells, the inhibitory effect persists for several hours, even after washing cells to remove away the unbound pseudopeptide. Under such conditions, the attachment of HIV particles to cells is inhibited as efficiently as by neutralizing monoclonal antibodies directed against the V3 loop. In view of its specific mode of action on various HIV isolates, HB-19 represents a potential anti-HIV drug.

The anti-HIV pseudopeptide HB-19 forms a complex with the cell-surface-expressed nucleolin independent of heparan sulfate proteoglycans.

The HB-19 pseudopeptide 5[Kpsi(CH(2)N)PR]-TASP, psi(CH(2)N) for reduced peptide bond, is a specific inhibitor of human immunodeficiency virus (HIV) infection in different CD4(+) cell lines and in primary T-lymphocytes and macrophages. Here, by using an experimental CD4(+) cell model to monitor HIV entry and infection, we demonstrate that HB-19 binds the cell surface and inhibits attachment of HIV particles to permissive cells. At concentrations that inhibit HIV attachment, HB-19 binds cells irreversibly, becomes complexed with the cell-surface-expressed nucleolin, and eventually results in its degradation. Accordingly, by confocal immunofluorescence microscopy, we demonstrate the drastic reduction of the cell-surface-expressed nucleolin following treatment of cells with HB-19. HIV particles can prevent the binding of HB-19 to cells and inhibit complex formation with nucleolin. Such a competition between viral particles and HB-19 is consistent with the implication of nucleolin in the process of HIV attachment to target cells. We show that another inhibitor of HIV infection, the fibroblast growth factor-2 (FGF-2) that uses cell-surface-expressed heparan sulfate proteoglycans as low affinity receptors, binds cells and blocks attachment of HIV to permissive cells. FGF-2 does not prevent the binding of HB-19 to cells and to nucleolin, and similarly HB-19 has no apparent effect on the binding of FGF-2 to the cell surface. The lack of competition between these two anti-HIV agents rules out the potential involvement of heparan sulfate proteoglycans in the mechanism of anti-HIV effect of HB-19, thus pointing out that nucleolin is its main target.

The V3 loop-mimicking pseudopeptide 5[Kpsi(CH2N)PR]-TASP inhibits HIV infection in primary macrophage cultures.

The V3 loop-mimicking pseudopeptide 5[Kpsi(CH2N)PR]-TASP [psi(CH2N) representing a reduced peptide bond], which presents pentavalently the tripeptide Kpsi(CH2N)PR, is a potent inhibitor of HIV entry. By its capacity to bind specifically protein components on the cell surface, 5[Kpsi(CH2N)PR]-TASP blocks the attachment of virus particles to permissive CD4+ cells. Here, the inhibitory effect of 5[Kpsi(CH2N)PR]-TASP was investigated in monocyte-derived macrophages (MDMs) infected by the monocytotropic HIV-1(Ba-L) isolate. We show that 5[Kpsi(CH2N)PR]-TASP inhibits HIV-1(Ba-L) infection in a dose-dependent manner, with more than 90% inhibition at 2 microM concentration. On the other hand, the control 5[QPQ]-TASP construct and the monovalent Kpsi(CH2N)PR tripeptide have no effect even at high concentrations. Under such experimental conditions, the biotin-labeled 5[Kpsi(CH2N)PR]-TASP, but not the Kpsi(CH2N)PR construct, binds specifically to the surface of MDMs and forms a stable complex with the cell surface-expressed nucleolin, as has been demonstrated to be the case in peripheral blood mononuclear cells. Infection of MDMs by HIV-1(Ba-L) could also be inhibited by beta-chemokines RANTES and MIP-1beta. Interestingly, association of low concentrations of 5[Kpsi(CH2N)PR]-TASP and beta-chemokines results in a synergistic inhibitory effect on HIV infection compared with the effect observed with each reagent alone. The inhibitory effect of 5[Kpsi(CH2N)PR]-TASP in primary macrophage cultures point out its potential as an anti-HIV drug in cells, which are the natural viral targets.

Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.