Soluble glycosaminoglycans Do not potentiate RANTES antiviral activity on the infection of primary macrophages by human immunodeficiency virus type 1.

Macrophages play an important role in human immunodeficiency virus (HIV)-1 infection. They exist in various differentiation and activation states in vivo, a heterogeneity that may affect their interactions with HIV-1 and susceptibility to drugs. Here, we found that RANTES and MIP-1beta, heparin, or soluble chondroitin sulfate B, but not chondroitin sulfate A, inhibited HIV-1(BaL) infection of macrophages obtained as the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2 days without either nonadherent PBMC or added cytokines (MDM-5d), whereas they did not affect infection of macrophages obtained as the adherent cells recovered from 1-h incubation of PBMC and subsequent 7-day culture with macrophage colony-stimulating factor (MDM-MCSF). Such different behavior was not related to differences in HIV-1 binding but rather to postbinding steps, as HIV-1(BaL) attached similarly to MDM-5d and MDM-MCSF, a binding that was affected by soluble glycosaminoglycans but not by RANTES. Of note, CCR5 expression on both types of MDM was comparable, and it was not downregulated by RANTES on either. Mixing RANTES with each of the glycosaminoglycans did not restore inhibition of MDM-MCSF infection by HIV-1; however, heparin at concentrations that had low antiviral activity for MDM-5d counteracted RANTES anti-HIV-1 activity for these cells, whereas chondroitin sulfate B had no additive effect on that of RANTES. Both glycosaminoglycans affected RANTES binding to MDM. Thus, in contrast to cell surface proteoglycans that contribute to the attachment of RANTES to macrophages and enhance its anti-HIV-1 activity, soluble glycosaminoglycans do not facilitate, and may even offset, the anti-HIV-1 activity of RANTES.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein.

Protein-protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the beta-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 microM IC(50). Structural analysis by NMR showed that both the backbone of the chimeric beta-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC(50) of 0.1-1.0 microM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4(+) cells by different virus isolates. Thus, core regions of large protein-protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein-protein interactions that may represent useful tools in biology and in drug discovery.

In-vivo delivery of therapeutic proteins by genetically-modified cells: comparison of organoids and human serum albumin alginate-coated beads.

We have designed a self-assembling multimeric soluble CD4 molecule by inserting the C-terminal fragment of the alpha chain of human C4-binding protein (C4bp alpha) at the C-terminal end of human soluble CD4 genes. This CD4-C4bp alpha fusion protein (sMulti-CD4) and two other reference molecules, a fusion protein of human serum albumin (HSA) and the first two domains of CD4 (HSA-CD4) and monomeric soluble CD4 (sMono-CD4), were delivered in vivo by genetically modified 293 cells. These cells were implanted in mice as organoids and also encapsulated in HSA alginate-coated beads. sMulti-CD4 showed an apparent molecular weight of about 300-350 kDa, in accordance with a possible heptamer formula. sMulti-CD4 produced either in cell culture or in vivo in mice appeared to be a better invitro inhibitor of HIV infection than sMono-CD4. Plasma levels of sMulti-CD4, HSA-CD4, and sMono-CD4 reached approximately 2,300, 2,700, and 170 ng/mL, respectively, 13 weeks after in-vivo organoid implantation, which had formed tumours at that time. This suggests that the plasma half-life of sMulti-CD4 is much longer than that of sMono-CD4. The 293 xenogeneic cells encapsulated in HSA alginate-coated beads remained alive and kept secreting sMono-CD4 or HSA-CD4 continuously at significant levels for 18 weeks in nude mice, without tumour formation. When implanted in immunocompetent Balb/c mice, they were rejected two to three weeks after implantation. In contrast, encapsulated BL4 hybridoma cells remained alive and kept secreting BL4 anti-CD4 mAb for at least four weeks in Balb/c mice. These results suggest the clinical potential of the C4bp-multimerizing system, which could improve both the biological activity and the poor in-vivo pharmacokinetic performance of a monomeric functional protein like soluble CD4. These data also show that a systemic delivery of therapeutic proteins, including immunoglobulins, can be obtained by the in-vivo implantation of engineered allogeneic cells encapsulated in HSA alginate-coated beads.

Synthetic full-length and truncated RANTES inhibit HIV-1 infection of primary macrophages.

OBJECTIVE: To determine the effect of beta-chemokines on HIV-1 infection of primary macrophages, and to search for chemokine derivatives devoid of biological effects but efficient at protecting CD4+ T lymphocytes and macrophages against HIV-1. DESIGN: Use of chemically synthesized molecules devoid of biological contaminants and monocyte-derived macrophages from healthy donors. METHODS: Full-length RANTES was chemically synthesized together with three derivatives, truncated of seven, eight and nine amino acids at the amino-terminus ([8-68]RANTES, [9-68]RANTES and [10-68]RANTES), which were tested for their biological activity and antiviral effects. RESULTS: Whereas full-length and truncated RANTES derivatives bound to beta-chemokine receptor CCR-5 with the same affinity as recombinant RANTES, the truncated forms were not chemotactic and acted as CCR-5 antagonists in this respect, although a partial agonist effect was noted on cell metabolism. Full-length RANTES and [8-68]RANTES protected T lymphocytes and macrophages from infection by HIV-1, although 10-fold higher concentrations of the truncated analogues were necessary to achieve the same effect as full-length RANTES. With regard to the effect of RANTES on HIV-1 infection of primary macrophages, our results contrast with most previously reported data. CONCLUSION: These data indicate that through binding to CCR-5, truncated RANTES derivatives that are devoid of detectable biological effects may represent candidates as drugs to protect both lymphocytes and macrophages from HIV- 1.

Effect of RANTES on the infection of monocyte-derived primary macrophages by human immunodeficiency virus type 1 and type 2.

The effect of beta chemokines on human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages is controversial, and their effect on HIV-2 infection of these cells has not yet been documented. We examined the effect of synthetic and recombinant regulated-on-activation, normal T cell-expressed and -secreted (RANTES) on HIV-1 and HIV-2 infection of primary monocyte-derived-macrophages (MDM) that were obtained as the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2-day culture without peripheral blood mononuclear cells (PBMCs) nor added cytokines. These MDM expressed CD4, CCR5 and CXCR4, the major coreceptors for HIV macrophage- and T cell-tropic isolates, respectively. Infection of MDM from different donors with HIV-1 or HIV-2 macrophage-tropic strains was reproducibly inhibited by RANTES. This inhibition depended on RANTES continuous presence in culture during and after infection. Treatment of MDM with RANTES just before or during, but not after, exposure to virus did not protect MDM from infection. When RANTES was added after MDM had been infected, and was continuously maintained in culture thereafter, no inhibition occurred and limited enhancement of infection could be observed. These data indicate that RANTES inhibits HIV-1 as well as HIV-2 infection of MDM, likely at a post-binding step, and support the role of CCR5 as the major coreceptor for HIV-1 and HIV-2 entry into primary macrophages.

The inhibitory effect of RANTES on the infection of primary macrophages by R5 human immunodeficiency virus type-1 depends on the macrophage activation state.

We investigated whether culture conditions could affect the RANTES antiviral effect on human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages. Monocyte-derived macrophages (MDM) were obtained either as (1) the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2 days without nonadherent PBMC or added cytokines (MDM-5d), or (2) as the adherent cells recovered from 1-h incubation of PBMC, which were cultured for 7 days with macrophage colony-stimulating factor (M-CSF; MDM-MCSF). Infection of MDM-5d from different donors with HIV-1 R5 strains was reproducibly inhibited by RANTES (IC50 < or = 10 nM), but infection of MDM-MCSF was not inhibited by > or = 100 nM RANTES, even when added at initiation of cultures, although it was still inhibited by a CD4 antibody. RANTES had no antiviral effect when MDM-5d were treated with physiological concentrations of M-CSF or GM-CSF before infection. CCR5 and CXCR4 expression as well as that of other cell surface molecules, including adhesion molecules, was not affected by the cytokines. MDM-MCSF from delta 32CCR5 homozygous individuals did not render them permissive to HIV-1, suggesting that it is unlikely that the virus uses another coreceptor. RANTES binding to MDM was chondroitin sulfate, but not heparan sulfate, dependent, and RANTES bound more efficiently to MDM-5d than to MDM-MCSF. Chondroitin sulfate removal partially offset the RANTES antiviral effect for MDM-5d. Thus RANTES anti-HIV-1 activity for primary macrophages depends on culture conditions and their consequent activation status, which may lead to differences in proteoglycan surface expression. These data may be relevant for the development of chemokine-based therapy for HIV-1 infection.

HIV type 1 V3 peptide constructs act differently on HIV type 1 infection of peripheral blood lymphocytes and macrophages.

We have previously shown that a multibranched peptide construct derived from the tip of the B clade V3 loop consensus sequence (MPBC1: [GPGRAF]8-[K]4-[K]2-K-beta A-OH), but not V3 monomer peptides, inhibit human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ T cells from immortalized lines. Here, we show that MBPC1 attaches to normal peripheral blood lymphocytes (PBLS) and monocytes but not to erythrocytes. While treatment with 5 microM MBPC1 had no significant antiviral effect on HIV-1Ba-L infection of monocyte-derived macrophages as assessed by p24 production in culture supernatants, this dose inhibited both HIV-1Ba-L and HIV-1LAI infection of PBLs. Virus production was inhibited up to 90% when MBPC1 was added to PBLs immediately after the virus, and was inhibited about 50% when it was added after 3 days; no effect was noted when it was added 7 days postinfection. MBPC1 did not affect PBL growth or IL-2 receptor and CD4 surface expression level. These observations suggest a selective antiviral effect of MBPC1 on CD4+ T lymphocytes and they provide additional circumstantial evidence that HIV-1 enters lymphocytes and monocytes by different mechanisms.

Antiviral activity of derivatized dextrans on HIV-1 infection of primary macrophages and blood lymphocytes.

The present study demonstrates at the molecular level that dextran derivatives carboxymethyl dextran benzylamine (CMDB) and carboxymethyl dextran benzylamine sulfonate (CMDBS), characterized by a statistical distribution of anionic carboxylic groups, hydrophobic benzylamide units, and/or sulfonate moieties, interact with HIV-1 LAI gp120 and V3 consensus clades B domain. Only limited interaction was observed with carboxy-methyl dextran (CMD) or dextran (D) under the same conditions. CMDBS and CMDB (1 microM) strongly inhibited HIV-1 infection of primary macrophages and primary CD4+ lymphocytes by macrophage-tropic and T lymphocyte-tropic strains, respectively, while D or CMD had more limited effects on M-tropic infection of primary macrophages and exert no inhibitory effect on M- or T-tropic infection of primary lymphocytes. CMDBS and CMDB (1 microM) had limited but significant effect on oligomerized soluble recombinant gp120 binding to primary macrophages while they clearly inhibit (> 50%) such binding to primary lymphocytes. In conclusion, the inhibitory effect of CMDB and the CMDBS, is observed for HIV M- and T-tropic strain infections of primary lymphocytes and macrophages which indicates that these compounds interfere with steps of HIV replicative cycle which neither depend on the virus nor on the cell.