Role of N-linked glycans in the interaction between the envelope glycoprotein of human immunodeficiency virus and its CD4 cellular receptor. Structural enzymatic analysis.

gp120 and CD4 are two glycoproteins that are considered to interact together to allow the binding of HIV to CD4+ cells. We have utilized enzymatic digestion by endoglycosidases in order to analyze N-linked carbohydrate chains of these proteins and their possible role in the interaction of gp120 or gp160 with CD4. SDS denaturation was not necessary to obtain optimal deglycosylation of either molecule, but deglycosylation of CD4, nonetheless, depended on the presence of 1% Triton X-100. Endo H and Endo F that cleave high mannose type and biantennary glycans diminish the molecular mass of the glycoproteins from 120 or 160 Kd to 90 or 130 Kd, respectively; but these enzymes had no action on CD4 glycans. Endo F N-glycanase mixture, which acts on all glycan species, including triantennary chains, led to complete deglycosylation of gp120/160 and of CD4. Therefore, probably half of the glycan moieties of gp120/160 are composed of high mannose and biantennary chains, the other half being triantennary species. The carbohydrate structures of CD4 seems to be triantennary chains. To analyze the binding of gp120/160 to CD4, we used a molecular assay in which an mAb (110-4) coupled to Sepharose CL4B allowed the attachment of soluble gp120/160 to the beads; 125I-sCD4 was then added to measure the binding of CD4 to different amounts of gp120/160. Binding to gp160 was not modified when using completely deglycosylated 125I-sCD4, while deglycosylation of gp120 or of gp160 resulted in the decrease of the binding to native CD4 by two- and fivefold, respectively. Native and deglycosylated gp120/160 bound to CD4+ cells with comparable affinities. In addition, deglycosylated gp120 displaced 125I-gp160 binding to CD4+ cells and inhibited fusion of fresh Molt-T4 cells with CEM HIV1- or HIV2-infected cells to the same extent. Taken together, these results indicate that carbohydrates of CD4 and of gp120/160 do not play a significant role in the in vitro interaction between these two molecules.

Loss of CD4 membrane expression and CD4 mRNA during acute human immunodeficiency virus replication.

Using mAbs and genomic probe to the CD4 molecule, the HIV receptor, we demonstrated that HIV replication induces the disappearance of its functional receptor from the cell surface by two distinct mechanisms. First, after being expressed onto the cell surface, HIV envelope gp110 will complex CD4, efficiently masking the CD4 epitope used by the virus to bind its receptor. This phenomenon occurs on the surface of each infected cell and is not due to the release of soluble gp110; infection with recombinant HIV/vaccinia viruses expressing a mutated HIV env gene designed to prevent gp110 release from the cell surface induces a similar gp/CD4 complexes formation. Second, virus replication induces a dramatic and rapid loss of CD4 mRNA transcripts, preventing new CD4 molecules from being synthesized. These two mechanisms of receptor modulation could have been developed to avoid reinfection of cells replicating the virus as well as to produce more infectious particles. These results suggest that the classical virus interference documented for other retroviruses might not only be due to receptor/envelope interaction, but might also depend on receptor gene expression.

Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes.

Lymphadenopathy associated virus ( LAV ) has been isolated from patients with the acquired immunodeficiency syndrome (AIDS) or lymphadenopathy syndrome. Since the immune deficiency in AIDS seems to be primarily related to the defect of the helper-inducer T lymphocyte subset, the possibility that LAV is selectively tropic for this subset was investigated. Fractionation of T lymphocytes was achieved by cellular affinity chromatography with monoclonal antibodies. In a hemophilic patient who was a healthy carrier of LAV , reverse transcriptase activity and virus particles detected by electron microscopy were found only in cultures of helper-inducer lymphocytes. When infected with LAV in vitro, lymphocyte subsets from normal individuals yielded similar results. Virus production was associated with impaired proliferation, modulation of T3-T4 cell markers, and the appearance of cytopathic effects. The results provide evidence for the involvement of LAV in AIDS.

Functions of HIV envelope glycans.

The unusually highly glycosylated state of the major envelope glycoprotein (gp160) of the human immunodeficiency virus has offered a challenge to both glycobiologists and virologists. What is the functional significance of such a mass of glycans and how might they be manipulated to disadvantage virus pathogenesis? Some answers to each of these questions have already been obtained: N-linked glycans are necessary for the creation, but not the maintenance, of a bioactive conformation, and drug-induced alteration of the glycosylation pattern can lead to impaired virus infectivity. As a model for studying glycan function and as a target for antiviral therapy, gp160 represents a unique candidate.

Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate.

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated Kd in the 10(-4) M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.

A monoclonal antibody directed to sulfatide inhibits the binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein to macrophages but not their infection by the virus.

We show here that human immunodeficiency virus (HIV) envelope glycoproteins (gp160/gp120) bind to sulfatide and galactosyl ceramide. By immunofluorescence labeling with monoclonal antibody (mAb) A2B5, specific for ganglioside/sulfatide, we detect negatively charged glycolipids on CD4+ cells of the macrophage lineage and lymphocytes. Labeling of monocyte-derived macrophages (MDM) with mAb A2B5 was reproducibly found in 29 healthy donors, independently of the culture method and duration up to 11 days. The binding of the mAb to neuraminidase-treated MDM was unchanged relative to control cells, but mAb binding decreased after arylsulfatase treatment, which indicates that MDM membrane sulfatide is its major ligand. Preincubating MDM with the mAb partially (40-60%) but significantly inhibited the binding of HIV-1LAI radiolabeled recombinant gp160 to the cells. Similarly, the mAb entailed limited (32%) but significant inhibition of gp160 binding to cells of the monocytic U937 line but not to lymphoid CEM cells. However, mAb A2B5 did not inhibit the infection of CEM nor of U937 cells by HIV-1LAI strain, nor of MDM by monocytotropic HIV-1BaL. Thus, although sulfatide may be involved in the binding of HIV env glycoprotein to MDM or monocytic U937 cells, this does not play a significant role in HIV infection of these CD4+ cells.

Involvement of the HIV-1 external envelope glycoprotein 120 (gp120) C2 region in gp120 oligomerization.

A synthetic peptide resembling the C2 region of human immunodeficiency virus type 1 (HIV-1) gp120 (C2-Lai: amino acids (aa) 273-288), inhibited (C50 = 200 microM) gp120 calcium-dependent binding of N-acetyl-beta-D-glucosaminyl and mannosyl residues exposed on natural glycoprotein bovine fetuin whereas a peptide derived from an aa sequence downstream of C2-Lai (C2-SC19) had no such effect (C50 > 1000 microM). No calcium-carbohydrate-specific binding of C2-Lai to fetuin was detected. In addition, C2-Lai was also found to inhibit the calcium-dependent oligomerization of gp120: while recombinant gp120 (rgp120) was recovered mainly as oligomers (78%) in 10 mM CaCl2, in contrast to 100% monomers in 1mM CaCl2, mostly monomers (67%) were found in 10 mM CaCl2 in the presence of C2-Lai. Peptide C2-SC19 and carbohydrate structures such as fetuin, fucoidin, dextran or mannan did not significantly affect gp120 oligomerization. Electrophoresis and gel filtration analysis also showed that C2-Lai aggregated in the form of 20 kDa compounds, which is compatible with association of 10 molecules. Taken together, these results demonstrate that the C2 domain is involved in gp120 oligomerization and suggest that gp120 oligomers but not monomers have specific carbohydrate binding properties.

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.

Lipopolysaccharide can block the potential of monocytes to differentiate into dendritic cells.

We examined whether priming monocytes (MO) with lipopolysaccharide (LPS) influenced their further differentiation into either macrophages (Mphi) or dendritic cells (DC). LPS-primed MO differentiated into Mphi when cultured further with Mphi colony-stimulating factor (M-CSF) but, if cultured then with granulocyte/Mphi (GM)-CSF and IL-4 (interleukin-4), only about 30% of the cells differentiated into CD1a+ CD14- DC and half became CD1a- CD14+ Mphi. Cytokines present during LPS priming could affect subsequent MO differentiation. Relative to priming with LPS alone, adding M-CSF to LPS did not modify differentiation of MO to Mphi in further culture with M-CSF, nor did it change the way of differentiation of MO into DC was altered if culture was later switched to GM-CSF/IL-4. Using GM-CSF/IL-4 plus LPS upon priming did not modify differentiation of MO to Mphi in further culture with M-CSF, as compared to priming with GM-CSF/IL-4 alone, but it counteracted the effect of LPS on the differentiation of MO to DC in further culture with GM-CSF/IL-4: about 75% of cells then became DC. Alternatively, despite activation by LPS, mature M-CSF-induced Mphi preserved the potential to differentiate into DC on subsequent culture with GM-CSF/IL-4. Thus, LPS, a bacterial product known to sustain maturation of MO/Mphi as well as of DC, may block the differentiation of MO into DC, except if signal triggering DC differentiation is delivered concomitantly, and modulate in this manner the induction of adaptive immune responses to infection.

IL-4 and CD40 ligation affect differently the differentiation, maturation, and function of human CD34+ cell-derived CD1a+CD14- and CD1a-CD14+ dendritic cell precursors in vitro.

We examined the effect of interleukin (IL)-4 or CD40 ligation on the differentiation and maturation of CD1a+CD14- and CD1a-CD14+ dendritic cell (DC) precursors. Cord blood CD34+ cells were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha), to which stem cell factor and Flt-3 ligand were added for 5 days. Phenotypic analysis of DC precursors on culture day 7 showed that CD1a+CD14- cells expressed higher CD11c and CD80 levels and lower CD116/GM-CSFR and CCR-5 levels than their CD1a-CD14+ counterparts. Culturing CD1a+CD14- precursors with GM-CSF and TNF-alpha resulted in DC with heterogeneous CD1a, HLA;SMDR (DR), CD11b, and CD83 expression, 10% of which acquired CD14. IL-4 and CD40 ligation affected their differentiation in contrasting ways: IL-4 induced CD1ahiCD14-DRloCD11b+CD83-S100+ DC with reduced MLR-stimulating capacity, whereas CD40 ligation led to CD1alo/-CD14-CD40-DRhiCD11b-CD83+S100+/- DC with stronger MLR-stimulating capacity. Also, both IL-4 and CD40 ligation promoted ReIB expression and nuclear translocation. When CD1a-CD14+ precursors were maintained in only the presence of GM-CSF and TNF-alpha, this led to mixed populations of adherent macrophages and nonadherent CD1a-CD14+ monocytes, and of CD1a+CD14- and CD1a+CD14+ DC, which were DRloCD11b+CD83-S100-. IL-4 or CD40 ligation prevented their differentiation into macrophages and resulted in DC with phenotypes close to those issued from CD1a+CD14- precursors, with only a minority staying CD14+ but most being S100-; their MLR-stimulating capacity also increased but remained lower than that of DC differentiated from CD1a+CD14- precursors. Thus, IL-4 or CD40 ligation induced CD1a+CD14- and CD1a-CD14+ DC precursors to differentiate into phenotypically close but functionally different DC populations, suggesting that DC function is primarily determined by their origin. The heterogeneity of DC should then be related to different developmental pathways and to different stages of maturation/activation.

Identification of mature and immature human thymic dendritic cells that differentially express HLA-DR and interleukin-3 receptor in vivo.

We have previously shown that thymic CD34+ cells have a very limited myeloid differentiation capacity and differentiate in vitro mostly into CD1a+-derived but not CD14+-derived dendritic cells (DC). Herein we characterized the human neonatal thymic DC extracted from the organ in relationship with the DC generated from CD34+ cells in situ. We show that in vivo thymic DC express E cadherin, CLA, CD4, CD38, CD40, CD44, and granulocyte-macrophage colony-stimulating factor-R (GM-CSF-R; CD116) but no CD1a. According to their morphology, functions, and surface staining they could be separated into two distinct subpopulations: mature HLA-DRhi, mostly interleukin-3-R (CD123)-negative cells, associated with thymocytes, some apoptotic, and expressed myeloid and activation markers but no lymphoid markers. In contrast, immature HLA-DR+ CD123hi CD36+ cells with monocytoid morphology lacked activation and myeloid antigens but expressed lymphoid antigens. The latter express pTalpha mRNA, which is also found in CD34+ thymocytes and in blood CD123hi DC further linking this subset to lymphoid DC. However, the DC generated from CD34+ thymic progenitors under standard conditions were pTalpha-negative. Thymic lymphoid DC showed similar phenotype and cytokine production profile as blood/tonsillar lymphoid DC but responded to GM-CSF, and at variance with them produced no or little type I interferon upon infection with viruses and did not induce a strict polarization of naive T cells into TH2 cells. Their function in the thymus remains therefore to be elucidated.

Evaluation of structure-antigenicity relationship of peptides from human immunodeficiency virus type 1 (HIV-1) p18 protein by circular dichroism.

The antigenicity of Human Immunodeficiency Virus type 1 (HIV-1) matrix p18 protein was evaluated by analyzing the specificity of anti-p18 antibodies elicited either in HIV-1 infected humans, or in HIV-1 infected or immunized chimpanzees, against a panel of long and short overlapping synthetic peptides [from 12 to 46 amino acid (aa) residues] covering the entire sequence of p18. The relationship between peptide structure and antigenicity was further investigated by probing the secondary structures of the peptides by circular dichroism. The results obtained clearly showed the immunodominance of the N-terminal region mimicked by peptide P1 (aa 2-45), which reacted with 52 and 100% of human and chimpanzee anti-p18 sera, respectively. In contrast smaller 15 aa long peptides C1, C2, C3, C4 and P3 which cover the entire sequence of immunodominant peptide P1, showed only weak or no reactivity. In contrast to widely accepted hypotheses, circular dichroism analysis of both small and large peptides secondary structures did not show any obvious correlation between antigenicity and the ability of peptides to adopt an ordered conformation.

Specificity of anti-P25 antibodies produced against whole HIV-1 particles or soluble forms of the protein.

Specificity of anti-p25 antibodies produced against either whole Human Immunodeficiency Virus type 1 (HIV-1) particles in humans and chimpanzees, or against soluble forms of the protein in chimpanzees and rabbits was analyzed by ELISA using a panel of 37 long (> or = 30 residues) or shorter (9-21 residues) overlapping peptides covering the entire p25 sequence. Antibodies elicited by intact virions presented similar reactivity patterns in HIV-1-infected humans and in HIV-1-infected or immunized chimpanzees and recognized only a limited region mostly the C-terminus of the molecule. Moreover, 8 of the human sera (36%), which nonetheless reacted with high titers and avidity with native p25, did not bind to any long or short peptide. These results suggest that the majority of antibodies elicited by viral particles are presumably directed to conformational epitopes. In contrast, antibodies raised against soluble forms of p25 could react against all long peptides but one (residues 211-245) and against some short peptides, indicating that most of p25 sequence may be immunogenic under these conditions. These results suggest that the reactivity spectrum of anti p25 antibodies is rather different if they are produced against intact HIV-1 particles or the soluble protein. They also indicate that it may be possible to manipulate the specificity of the humoral immune response by using either intact virions or purified proteins.

The influence of interleukin (IL)-4, IL-13, and Flt3 ligand on human dendritic cell differentiation from cord blood CD34+ progenitor cells.

Culturing cord blood CD34+ cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha for 12 days, and stem cell factor (SCF) for 5 days, resulted in a 40- +/- 26-fold expansion in cell numbers, with 38 +/- 20% dendritic cells (DCs). Interleukin (IL)-4 and IL-13, which share properties, were examined first. Adding either one to the former baseline condition beginning on day 0 halved cell growth while the percentage of DCs increased to 60-70%, resulting in unchanged DC yields. Delaying use of IL-4 or IL-13 to day 5 led to 25-fold cell expansion with approximately 80% DC, the yield of which was then twofold over that of baseline control cultures, while numbers of other cells decreased. IL-4 and IL-13 had no additive or antagonistic effect on DC generation. The effect of Flt3 ligand (FL), known to enhance proliferation of hematopoietic progenitors induced by other growth factors, was examined next. FL added alone induced DC in the same manner as SCF. Using both FL and SCF throughout the culture period enhanced total cell recovery fourfold above that of baseline control cultures on day 12 compared with > or =2.5-fold if either one was stopped on day 5. When both FL and SCF were used for 12 days, DC recovery was fivefold that of control cultures, whereas it was to three- to 3.5-fold when either one was stopped on day 5. A similar trend was noted for CD15+ cells, and, to a lesser extent, for CD14+ cells. Finally, using SCF and FL for 12 days, with IL-4 or IL-13 added from day 5 onwards, led to comparably enhanced cell yields relative to control cultures with approximately 60% DC. These data underline the need to use appropriate cytokine combinations and schedules to optimize generation of DCs from CD34+ progenitors. Associated with GM-CSF and TNF-alpha, IL-4 or IL-13 promotes differentiation and maturation of DCs over other myeloid cells. Under the same baseline conditions, FL appears to potentiate SCF throughout the culture period, inducing proliferation and development of DC as well as of other myeloid cells.

Detection of anti-CD4 autoantibodies in the sera of HIV-infected patients using recombinant soluble CD4 molecules.

The use of purified recombinant soluble CD4 (sT4) allowed the detection of high titers of anti-CD4 immunoglobulins in the sera of three out of 33 HIV-infected patients. Binding of these antibodies to sT4 was first detected by enzyme-linked immunosorbent assay (ELISA), and their reactivity in the assay was blocked in a dose-dependent manner by preincubation with sT4. The antibodies could also immunoprecipitate iodinated sT4, but they failed to recognize CD4 expressed on the surface of CD4+ lymphocytes or cell lines. An ELISA which used as an antigen a truncated soluble CD4 molecule containing only the first two amino-terminal domains of the CD4 molecule did not react with these sera in ELISA, nor did it block antibody binding to sT4. Both these human sera and Leu3a, a mouse monoclonal antibody (mAb) which recognizes an epitope of CD4 close to the HIV binding site, failed to compete with one another for binding to sT4. Because these antibodies did not recognize epitope(s) of the CD4 molecule close to the HIV binding site, they are not likely to be anti-idiotypic antibodies directed against anti-HIV envelope antibodies. The exact location of the recognized epitope(s), as well as the role, if any, of these antibodies in the pathophysiology of HIV infection remains to be determined.

Antibody-dependent cellular cytotoxicity against HIV-1 in sera of immunized chimpanzees.

After immunization of chimpanzees against HIV antigens, antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) were evaluated and compared with anti-HIV-antibody levels detected by enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody titers. Adult chimpanzees were immunized with different HIV-1 (LAV-BRU) antigen preparations: recombinant vaccinia virus (rVV) expressing gp160, p25 or p27nef; formalin- and beta-propiolactone-inactivated whole virus (inHIV); soluble recombinant gp160 either associated or not associated with other HIV proteins; a 25-mer peptide from the V3 region of gp120 coupled with KLH (V3-KLH). Immunization with the various rVV mixtures induced no or borderline ADCC increase above preimmune serum levels. Stronger and more sustained reactivity was elicited by inHIV. Purified HIV antigens elicited ADCC activity when the chimpanzees were naive; ADCC increased or remained at the same level when the animals had been preimmunized with rVV and/or inHIV. This type of reactivity apparently did not depend on whether gp160 alone or mixed with other proteins was used for immunization. The injection of V3-KLH resulted in only little, if any, recall ADCC response. ELISA antibody titers significantly correlated with ADCC and neutralizing antibody titers, but serum ADCC was independent of neutralizing antibody titers, an indication that the two latter serum activities are mediated by independent antibodies. Therefore, ADCC is elicited in the same manner as other antibody activities by the immunization of chimpanzees with inHIV or with purified recombinant HIV antigen preparations. The results obtained from the three chimpanzees of this series, which were subsequently challenged with infectious virus through the intravenous route, suggest that serum ADCC may be considered for vaccination purposes.

Monoclonal antibodies to the human immunodeficiency virus p18 protein cross-react with normal human tissues.

The immunohistochemical reactivity of four monoclonal antibodies (MAbs): CVK, 49-5, 49-6 and 63-FH2, raised against the p18 protein of HIV-1 was assessed in tissues obtained from HIV-infected and uninfected individuals. As already reported, all the MAbs specifically labelled follicular dendritic cells (FDC) in lymph nodes from HIV-infected patients with lymphadenopathy, and cells of microglial nodules in the brain from patients with AIDS-related encephalopathy. However, cross-reactivity with normal uninfected tissues was also observed: epithelial cells of the skin, the thymus and tonsils with CVK, and astrocytes in the brain of 49-6 and 63-FH2. Such cross-reactivities suggest that 'molecular mimicry' could exist between p18 of HIV and normal constituents of human cells. This phenomenon could be relevant for the diagnostic use of anti-p18 MAbs on pathological specimens, and it could be of importance in the pathophysiology of HIV infection.

HIV infection of monocytic cells: rôle of antibody-mediated virus binding to Fc-gamma receptors.

We investigated whether human immunoglobulin G (IgG) directed to gp110 may serve as an attachment system to Fc-gamma receptors (Fc-gamma R), allowing eventual infection of cells of the macrophage lineage. An anti-HIV IgG preparation that prevented viral particles and soluble recombinant radiolabelled envelope precursor gp160 from binding to CD4 on CEM lymphoid cells, and that strongly inhibited infection of these cells by HIV, was selected. In contrast, anti-HIV IgG, whether or not previously complexed to viral particles, bound to monocytic U937 cells that express both high Fc-gamma RI and low affinity Fc-gamma RII receptors. Precoating these cells with anti-HIV IgG or complexing the antibodies with soluble 125I-gp160 resulted in increased fixation of gp160 to the cells, which was inhibited by aggregated human normal IgG. These data indicate that anti-HIV IgG-dependent attachment of gp160 to monocytic cells occurs through both types of Fc-gamma R. In addition, this method of attachment resulted in productive infection of U937 cells that, since it was blocked in the presence of Leu3a, still appeared to involve gp110-CD4 interaction. Only slight enhancement of infectivity, such as described for other enveloped viruses, was noted, even when antibody concentration was titrated down. This mechanism may be one of the explanations why the humoral response to HIV is not usually protective.

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.

A molecular mechanism of inhibition of HIV-1 binding to CD4+ cells by monoclonal antibodies to gp110.

We have investigated the possible involvement in the interaction between HIV gp110 and its CD4 receptor of epitopes different from the currently known binding site(s) of the molecule. Four monoclonal antibodies (MAbs) to gp110 were used (Genetic Systems Corporation, Seattle, Washington, USA): one (110-1) recognized a peptide corresponding to the C-terminal part of gp110 (494-517); the other three (110-3, 110-4, 110-5) recognized the same peptide located at position 308-328. HIV or purified gp110 obtained from a vaccinia recombinant (Transgene S.A., Strasbourg, France) were pre-incubated with the MAb prior to addition to CD4+ cells. Specific binding of viral particles or of the soluble molecule was then determined by flow cytometer analysis, compared with that of control preparations where the MAb was added after HIV or gp 110 had been allowed to bind CD4+ cells. Significant inhibition of HIV binding was noted with the three MAbs to peptide (308-328), but not with 110-1. At the molecular level, these same MAbs decreased the affinity of interaction between CD4 and soluble gp110, although they could still label the latter molecule after it had bound to CD4+ cells. Therefore, steric hindrance may account for neutralization of HIV binding by antibodies that are actually directed to epitopes topographically distinct from the site of binding of gp110 to CD4.