anti-CCR5 Monoclonal Antibody Research Articles

A Predictive Model for Identifying the Most Effective Anti-CCR5 Monoclonal Antibody

CCR5 (R5) inhibition is increasingly being studied for its potential to prevent, treat, and cure illnesses. R5 is a transmembrane protein that interacts with the CD4 receptor and CXCR4 (X4) of T cells, allowing the attachment of HIV viruses to lymphocytes. Consequently, because R5 inhibition has performed well as a medicinal drug, such as maraviroc, many researchers have speculated that R5 inhibition via binding antibodies may effectively treat HIV patients. However, currently, there is a lack o

Targeted Immuno-Antiretroviral to Promote Dual Protection against HIV: A Proof-of-Concept Study.

The C–C motif chemokine receptor-5 (CCR5) expression on the T-cell surface is the prime barrier to HIV/AIDS eradication, as it promotes both active human immunodeficiency virus (HIV)-infection and latency; however, antiretrovirals (ARVs) suppress plasma viral loads to non-detectable levels. Keeping this in mind, we strategically designed a targeted ARVs-loaded nanoformulation that targets CCR5 expressing T-cells (e.g., CD4+ cells). Conceptually, CCR5-blocking and targeted ARV delivery would be a dual protection strategy to prevent HIV infection. For targeting CCR5+ T-cells, the nanoformulation was surface conjugated with anti-CCR5 monoclonal antibodies (CCR5 mAb) and loaded with dolutegravir+tenofovir alafenamide (D+T) ARVs to block HIV replication. The result demonstrated that the targeted-ARV nanoparticle’s multimeric CCR5 binding property improved its antigen-binding affinity, prolonged receptor binding, and ARV intracellular retention. Further, nanoformulation demonstrated high binding affinity to CCR5 expressing CD4+ cells, monocytes, and other CCR5+ T-cells. Finally, the short-term pre-exposure prophylaxis study demonstrated that prolonged CCR5 blockage and ARV presence further induced a “protective immune phenotype” with a boosted T-helper (Th), temporary memory (TM), and effector (E) sub-population. The proof-of-concept study that the targeted-ARV nanoformulation dual-action mechanism could provide a multifactorial solution toward achieving HIV “functional cure.”

CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties

BackgroundChemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. MethodsWe investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. ResultsTwo commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. ConclusionsCCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. General significanceWe provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

Several Commercially Available Anti-CCR5 Monoclonal Antibodies Lack Specificity and Should Be Used with Caution

CCR5 (CD195) is a receptor for the chemokines RANTES, MIP-1α, and MIP-1β and is used by HIV-1 as a co-receptor for entry into macrophages and CD4+ T cells. CCR5 exists in multiple conformations in the membrane and is present at low levels on human macrophages, making it difficult to detect. Nine commercially available anti-CCR5 monoclonal antibodies were evaluated for their specificity and their recognition of CCR5 expressed by macrophages. Unexpectedly, we found that three of the nine clones tested displayed substantial background binding to CCR5 negative cells, suggesting that these antibodies may give unreliable results. We recommend the use of clones CTC8, 45531 (PE conjugated), and 45523 and advise that data obtained using CTC5, 45531 (unconjugated), and 45549 anti-CCR5 antibody clones should be interpreted with care.

CCR5 blockade is well tolerated and induces changes in the tissue distribution of CCR5+ and CD25+ T cells in healthy, SIV-uninfected rhesus macaques

CCR5 is a main co-receptor for HIV, but also homes lymphocytes to sites of inflammation. We hypothesized that inhibition of CCR5 signaling would reduce HIV-associated chronic immune activation. To test this hypothesis, we administered an antagonistic anti-CCR5 monoclonal antibody (HGS101) to five uninfected rhesus macaques (RMs) and monitored lymphocyte dynamics in blood and tissue. CCR5 blockade resulted in decreased levels of CCR5+ T cells in blood and, at later timepoints, in lymph nodes. Additionally, the levels of CD25+ T cells increased in lymph nodes, but decreased in blood, bone marrow, and rectal mucosa. Finally, a profile of gene expression from HGS101-treated RMs revealed a subtle, but consistent, in vivo signature of CCR5 blockade that suggests a mild immune-modulatory effect. Treatment with anti-CCR5 antibody induces changes in the tissue distribution of CCR5+ and CD25+ T cells that may impact on the overall levels of immune activation during HIV and SIV infection.

Direct observation of chemokine receptors 5 on T-lymphocyte cell surfaces using fluorescent metal nanoprobes 2: Approximation of CCR5 populations

Metal nanoparticle probes were used as molecular imaging agents to detect the expression levels and spatial distributions of the CCR5 receptors on the cell surfaces. Alexa Fluor 647-labeled anti-CCR5 monoclonal antibodies (mAbs) were covalently bound to 20 nm silver nanoparticles to synthesize the mAb–metal complexes. We measured the single nanoparticle emission of the mAb–metal complexes, showing that the complexes displayed enhanced intensities and reduced lifetimes in comparison with the metal-free mAbs. Six HeLa cell lines with various CCR5 expressions were incubated with the mAb–metal complexes for the target-specific binding to the cell surfaces. Fluorescence cell images were recorded on a time-resolved confocal microscope. The collected images expressed clear CCR5 expression-dependent optical properties. Two regression curves were obtained on the basis of the emission intensity and lifetime over the entire cell images against the number of the CCR5 expression on the cells. The emission from the single mAb–metal complexes could be distinctly identified from the cellular autofluorescence on the cell images. The CCR5 spatial distributions on the cells were analyzed on the cell images and showed that the low-expression cells have the CCR5 receptors as individuals or small clusters but the high expression cells have them as the dense and discrete clusters on the cell surfaces.

The Lupane-type Triterpene 30-Oxo-calenduladiol Is a CCR5 Antagonist with Anti-HIV-1 and Anti-chemotactic Activities

The existence of drug-resistant human immunodeficiency virus (HIV) viruses in patients receiving antiretroviral treatment urgently requires the characterization and development of new antiretroviral drugs designed to inhibit resistant viruses and to complement the existing antiretroviral strategies against AIDS. We assayed several natural or semi-synthetic lupane-type pentacyclic triterpenes in their ability to inhibit HIV-1 infection in permissive cells. We observed that the 30-oxo-calenduladiol triterpene, compound 1, specifically impaired R5-tropic HIV-1 envelope-mediated viral infection and cell fusion in permissive cells, without affecting X4-tropic virus. This lupane derivative competed for the binding of a specific anti-CCR5 monoclonal antibody or the natural CCL5 chemokine to the CCR5 viral coreceptor with high affinity. 30-oxo-calenduladiol seems not to interact with the CD4 antigen, the main HIV receptor, or the CXCR4 viral coreceptor. Our results suggest that compound 1 is a specific CCR5 antagonist, because it binds to the CCR5 receptor without triggering cell signaling or receptor internalization, and inhibits RANTES (regulated on activation normal T cell expressed and secreted)-mediated CCR5 internalization, intracellular calcium mobilization, and cell chemotaxis. Furthermore, compound 1 appeared not to interact with beta-chemokine receptors CCR1, CCR2b, CCR3, or CCR4. Thereby, the 30-oxo-calenduladiol-associated anti-HIV-1 activity against R5-tropic virus appears to rely on the selective occupancy of the CCR5 receptor to inhibit CCR5-mediated HIV-1 infection. Therefore, it is plausible that the chemical structure of 30-oxo-calenduladiol or other related dihydroxylated lupane-type triterpenes could represent a good model to develop more potent anti-HIV-1 molecules to inhibit viral infection by interfering with early fusion and entry steps in the HIV life cycle.

Retracted: CCR5 blockade in combination with rapamycin prolongs cardiac allograft survival in mice

Summary Both chemokine receptor 5 (CCR5) blockade and rapamycin (rapa) are effective in modulating transplant immunity and led to prolonged allograft survival, yet a great many grafts were ultimately lost to acute rejection. In this study we examined the inhibition of CCR5 in combination with the treatment with rapa in cardiac transplantation. Fully major histocompatibility complex-mismatched murine cardiac allograft models were randomized to five groups. They were administered with anti-CCR5 antibody or control antibody and rapa or phosphate-buffered saline (PBS), respectively. An additional group was treated with anti-CCR5 antibody, rapa and anti-CD25 antibody. Allograft rejection was investigated by flow cytometric analyses and enzyme-linked immunospot assay. Allografts treated with anti-CCR5 antibody plus rapa showed significantly prolonged survival (83 ± 3 days, P < 0·001) compared with control antibody plus PBS-treated allografts (6 ± 1 days). Treatment with anti-CCR5 monoclonal antibody (mAb) plus rapa inhibited significantly the progression of chronic rejction. Further analysis of donor hearts in the anti-CCR5 antibody plus rapa-treated group demonstrated increased infiltration of CD4+CD25+forkhead box P3+ regulatory T cells, and depletion of CD25+ cells resulted in acute rejection of allografts in 18 ± 1 day. CCR5 blockade in combination with rapa is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated by CD25+ T cell recruitment and control of T lymphocyte proliferation.

CCR5 as Target for HIV-1 Gene Therapy

Acquired immune deficiency syndrome (AIDS) is caused by a lentivirus, human immunodeficiency virus type-1 (HIV-1). Viral entry is mediated by specific interaction of the viral envelope (Env) glycoprotein with a cell surface molecule CD4 which serves as the primary receptor and a chemokine (C-C or C-X-C motif) receptor CCR5 or CXCR4 which serves as a co-receptor. The viral Env, the cellular CD4 receptor, or the CCR5/CXCR4 co-receptors may be the targets of therapeutic interventions. Compared to the high variability of the viral Env protein, lack of variability in the CD4 receptor and the CCR5 or CXCR4 co-receptor makes them better targets to prevent viral entry. Downregulation of CD4 or CXCR4 is likely to have harmful consequences for the immune function or cellular maturation and homing. In contrast, individuals who lack functional CCR5 have no apparent immune defects, and show decreased susceptibility to HIV-1 infection and delayed progression to AIDS. CCR5 is essential for HIV-1 infection through all routes of transmission. Therefore, its downregulation may not only prevent disease progression, but also the spread of HIV-1 transmission. To block CCR5 function, a number of molecules were developed, including low molecular weight compounds, chemokines, N-terminally-modified chemokine analogues, chemokine-derived molecules, chemokine-based synthetic peptides, and anti-CCR5 monoclonal antibodies. Gene therapy strategies were developed using intrakines and intrabodies to prevent cell surface expression of CCR5 and zinc finger-nucleases, or using small interfering RNAs, antisense RNAs, or ribozymes to decrease co-receptor synthesis. This review describes the importance of targeting CCR5 and summarizes the status of various anti-CCR5 gene therapy strategies.

Gp120 V3-dependent Impairment of R5 HIV-1 Infectivity Due to Virion-incorporated CCR5

Entry of R5 human immunodeficiency virus type 1 (HIV-1) into target cells requires sequential interactions of the envelope glycoprotein gp120 with the receptor CD4 and the coreceptor CCR5. We investigated replication of 45 R5 viral clones derived from the HIV-1JR-FLan library carrying 0-10 random amino acid substitutions in the gp120 V3 loop. It was found that 6.7% (3/45) of the viruses revealed >or=10-fold replication suppression in PM1/CCR5 cells expressing high levels of CCR5 compared with PM1 cells expressing low levels of CCR5. In HIV-1V3L#08, suppression of replication was not associated with entry events and viral production but with a marked decrease in infectivity of nascent progeny virus. HIV-1V3L#08, generated from infected PM1/CCR5 cells, was 98% immunoprecipitated by anti-CCR5 monoclonal antibody T21/8, whereas the other infectious viruses were only partially precipitated, suggesting that incorporation of larger amounts of CCR5 into the virions caused impairment of viral infectivity in HIV-1V3L#08. The results demonstrate the implications of an alternative influence of CCR5 on HIV-1 replication.

CD4 Interacts Constitutively with Multiple CCR5 at the Plasma Membrane of Living Cells

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-microm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.

Increased expression of CCR5 in experimental autoimmune myocarditis and reduced severity induced by anti-CCR5 monoclonal antibody

Experimental autoimmune myocarditis (EAM) is a T-cell-mediated autoimmune disease. CCR5, which is expressed mostly on activated T cells and monocytes/macrophages, are potent chemotactic factors for autoimmune myocarditis. We investigated the role of CCR5 in the formation of experimental autoimmune myocarditis. Expression of CCR5 and its cognate ligands was assessed by RT–PCR and immunohistochemical analysis. Single-cell suspension of splenocytes and whole blood specimens from EAM mice were subjected to flow-cytometry analysis. We investigated the critical role of CCR5 in EAM mice by adoptively transferring CCR5-positive/negative T cells to mice and by neutralizing CCR5 with monoclonal antibody to observe the influence on the severity and prevalence of myocarditis. In this report, we found that CCR5-positive cells predominate in infiltrated inflammatory cells in cardiac tissue of EAM mice and CCR5-positive T cells in peripheral blood increased markedly in EAM mice compared with controls. Moreover, we demonstrated that the severity of myocarditis was significantly reduced when CCR5-negative T cells from EAM mice were adoptively transferred. When administrated with CCR5-positive T cells, the myocarditis was significantly aggravated. We also demonstrated that blockade of CCR5 with monoclonal antibodies significantly reduced severity of myocarditis in EAM mice. Overall, these findings indicate that CCR5 is important in the induction of EAM and inhibition of CCR5 with monoclonal antibody significantly reduces the severity of myocarditis. CCR5 may have the potential to become a new therapy target against autoimmune myocarditis.

Identification of human monoclonal antibodies specific for CCR5 from an antibody library derived from HIV-infected long-term non-progressors

It has been reported that about one quarter of long-term nonprogressors had anti-CCR5 antibodies and such antibodies were not found in disease progressing HIV-1 positive individuals [1]. To identify such antibodies we panned an HIV immune library constructed from bone marrow of three long-term nonprogressors against a synthetic sulfated N-terminal CCR5 peptide (2–15) (R5Nt). Twenty anti-CCR5 monoclonal antibodies were selected that bind to the R5Nt and cell-associated CCR5. Sequence analysis revealed that eighteen clones have VHs that were derived from the same germline sequence. Preliminary data showed that these antibodies inhibited primary HIV-1 isolates from clade B and E as tested in a pseudovirus assay. Identification of these anti-CCR5 human monoclonal antibodies indicates possible contribution to mechanisms determining the slow progression or lack of disease in long-term nonprogresssors. The selected anti-CCR5 antibodies may have potentials as therapeutics and/or prophylactics in combination with other HIV-1 neutralizing antibodies and antiretroviral drugs.

Resistance profile of a neutralizing anti-HIV monoclonal antibody, KD-247, that shows favourable synergism with anti-CCR5 inhibitors

The high-affinity humanized monoclonal antibody (MAb) KD-247 reacts with a tip region in gp120-V3 and cross-neutralizes primary isolates with a matching neutralization sequence motif. We induced an HIV-1 variant that was resistant to KD-247 by exposing the JR-FL virus to increasing concentrations of KD-247 in PM1/CCR5 cells, which expressed high levels of CCR5 in vitro. We determined the amino acid sequence of the gp120-encoding region of the JR-FL escape mutant from KD-247. To confirm that this substitution was responsible for the KD-247-resistance, a single-round replication assay was performed. We further evaluated the anti-HIV-1 interactions between KD-247 and various CCR5 inhibitors in vitro. At passage 8 of the culture in the presence of 1000 mug/ml KD-247, one amino acid substitution, Gly to Glu at position 314 (G314E), was identified in the V3-tip of gp120. A pseudotyped virus with the G314E mutation was highly resistant to KD-247. Unexpectedly, this mutant virus was sensitive to CCR5 inhibitors, RANTES, recombinant human soluble CD4 (rsCD4) and an anti-CCR5 MAb, but resistant to an anti-CD4 MAb, compared with the wild-type virus. We also found that combinations of KD-247 and CCR5 inhibitors were highly synergistic. The present data suggest that KD-247 has certain advantages for possible passive immunotherapy. They are: high concentrations of KD-247 are needed for viral acquisition of KD-247 resistance; the escape variants are more sensitive to CCR5 inhibitors and rsCD4; and there are high levels of synergism between KD-247 and CCR5 inhibitors at all concentrations tested.

The strength of the chemotactic response to a CCR5 binding chemokine is determined by the level of cell surface CCR5 density

We have shown that the intensity of expression of the C-C chemokine receptor CCR5 at the single CD4(+) cell level strongly determines the efficiency of its function as a coreceptor for human immunodeficiency virus type 1. By analogy, we examined if the number of CCR5 molecules at the cell surface might determine its chemotactic response to CCR5 ligands. To test this hypothesis, we measured by flow cytometry the migration of primary human T cells towards the CCR5-binding chemokine CCL5 in vitro. First, we observed a dose-dependent blockage of this migration exerted by an anti-CCR5 monoclonal antibody. Second, we sorted peripheral blood mononuclear cells into five subpopulations expressing various cell surface CCR5 densities, and observed a correlation between the intensity of migration towards CCL5 and the level of CCR5 expression on these subpopulations. Third, we transduced CCR5(+) peripheral blood mononuclear cells with the CCR5 gene, and observed that the CCR5 over-expression induced an over-migration towards CCL5. Finally, we observed in healthy donors a correlation between the chemotactic response of peripheral blood CD8(+) T cell to CCL5 and their level of surface CCR5 expression. T-cell surface CCR5 density, which is constant over time for a given individual, but varies drastically among individuals, might therefore be an important personal determinant of T-cell migration in many biological situations where CCR5-binding chemokines play a role, such as graft rejection, T helper 1-mediated auto-immune diseases, and infectious diseases involving CCR5. Moreover, our data highlight the therapeutic potential of CCR5 antagonists in these situations.

Expression Levels of CXC Chemokine Receptors 3 Are Associated with Clinical Phenotype of Type 1 Diabetes

Type 1 diabetes is recognized as one of T helper 1 cell (Th1)-mediated diseases. The purpose of this article was to investigate the expression levels of CXC chemokine receptor 3 (CXCR3) and CC chemokine receptor 5 (CCR5) on CD4 T cells as Th1 markers in Japanese patients with type 1 diabetes and control subjects. A total of 72 patients with type 1 diabetes and 24 healthy subjects were enrolled. Their peripheral mononuclear cells were obtained and stained with anti-CXCR3, anti-CCR5, and anti-CD4 monoclonal antibodies. Flow-cytometric analysis was performed and patients were classified according to their onset pattern as fulminant, typical, or slow onset. Statistical analysis was performed using ANOVA. CXCR3 expression on CD4 T cells in patients with a fulminant pattern of onset was significantly lower than that in the other groups, and that in patients with a typical pattern of onset was significantly higher than that in the other groups. CCR5 expression on CD4 T cells was not different among the three clinical phenotypes. CXCR3 expression level is associated with the onset pattern of type 1 diabetes. Further studies are needed to clarify the role of chemokines in type 1 diabetes.

An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains.

Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.

Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies.

CC-chemokine receptor 5 (CCR5) is the principal coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have generated a set of anti-CCR5 monoclonal antibodies and characterized them in terms of epitope recognition, competition with chemokine binding, receptor activation and trafficking, and coreceptor activity. MC-4, MC-5, and MC-7 mapped to the amino-terminal domain, MC-1 to the second extracellular loop, and MC-6 to a conformational epitope covering multiple extracellular domains. MC-1 and MC-6 inhibited regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory polypeptide-1beta, and Env binding, whereas MC-5 inhibited macrophage inflammatory polypeptide-1beta and Env but not RANTES binding. MC-6 induced signaling in different functional assays, suggesting that this monoclonal antibody stabilizes an active conformation of CCR5. Flow cytometry and real-time confocal microscopy showed that MC-1 promoted strong CCR5 endocytosis. MC-1 but not its monovalent isoforms induced an increase in the transfer of energy between CCR5 molecules. Also, its monovalent isoforms bound efficiently, but did not internalize the receptor. In contrast, MC-4 did not prevent RANTES binding or subsequent signaling, but inhibited its ability to promote CCR5 internalization. These results suggest the existence of multiple active conformations of CCR5 and indicate that CCR5 oligomers are involved in an internalization process that is distinct from that induced by the receptor's agonists.

Bovine immunodeficiency virus expression in vitro is reduced in the presence of beta-chemokines, MIP-1alpha, MIP-1beta and RANTES.

The inhibition of HIV expression in vitro by a cocktail of the beta-chemokines MIP-1alpha, MIP-1beta and RANTES provided the initial evidence that HIV utilizes chemokine receptors as co-receptors for infection of cells. Bovine immunodeficiency virus (BIV), a lentivirus, infects a wide variety of leukocyte populations, but the cellular receptor(s) utilized by this virus for infection of cells is not known. The purpose of this study was to determine whether MIP-1alpha, MIP-1beta and RANTES affect BIV expression in vitro, as a prelude to identifying the cellular receptors utilized by this virus. Fetal bovine lung (FBL) cells were pretreated with serial dilutions of a cocktail of the chemokines, and then the cells were infected with BIV. Virus expression in these cells was determined by counting the syncytia that had developed in the cultures by five days after infection. A significant decrease in syncytium formation, corresponding to increasing concentrations of the chemokines, was the result. Reacting the chemokines with chemokine-specific neutralizing antibodies prior to treatment of the cells neutralized the effect of the chemokines on virus replication in a dose-dependent manner, restoring viral expression to a level similar to that of untreated cells. The presence of a CCR5 homologue on the surface of FBL cells was confirmed using an anti-CCR5 monoclonal antibody and FACS analysis. Collectively, these data provide preliminary evidence that BIV may utilize the CCR5 receptor for infection of cells in vitro, but additional studies are necessary to confirm this.