The beta-chemokines MIP-1alpha, MIP-1beta and RANTES inhibit infection of CD4+ T cells by primary, non-syncytium-inducing (NSI) HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4+ T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of beta-chemokines. Expression of the beta-chemokine receptor CC-CKR-5 in CD4+, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a second receptor for NSI primary viruses.

The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. AbstractBackground: Dupilumab, a fully human monoclonal antibody that binds IL-4Rα and inhibits signaling of both IL-4 and IL-13, has shown efficacy across multiple diseases with underlying type 2 signatures and is approved for treatment of asthma, atopic dermatitis, and chronic sinusitis with nasal polyposis. We sought to provide a comprehensive analysis of the redundant and distinct roles of IL-4 and IL-13 in type 2 inflammation and report dupilumab mechanisms of action.Methods: Using primary cell assays and a mouse model of house dust mite-induced asthma, we compared IL-4 vs IL-13 vs IL-4Rα blockers.Results: Intranasal administration of either IL-4 or IL-13 confers an asthma-like phenotype in mice by inducing immune cell lung infiltration, including eosinophils, increasing cytokine/chemokine expression and mucus production, thus demonstrating redundant functions of these cytokines. We further teased out their respective contributions using human in vitro culture systems. Then, in a mouse asthma model by comparing in head-to-head studies, either IL-4 or IL-13 inhibition to dual IL-4/ IL-13 inhibition, we demonstrate that blockade of both IL-4 and IL-13 is required to broadly block type 2 inflammation, which translates to protection from allergeninduced lung function impairment. Notably, only dual IL-4/IL-13 blockade prevented eosinophil infiltration into lung tissue without affecting circulating eosinophils, demonstrating that tissue, but not circulating eosinophils, contributes to disease pathology.

CCR5 serves as a requisite fusion coreceptor for clinically relevant strains of human immunodeficiency virus type 1 (HIV-1) and provides a promising target for antiviral therapy. However, no study to date has examined whether monoclonal antibodies, small molecules, or other nonchemokine agents possess broad-spectrum activity against the major genetic subtypes of HIV-1. PRO 140 (PA14) is an anti-CCR5 monoclonal antibody that potently inhibits HIV-1 entry at concentrations that do not affect CCR5's chemokine receptor activity. In this study, PRO 140 was tested against a panel of primary HIV-1 isolates selected for their genotypic and geographic diversity. In quantitative assays of viral infectivity, PRO 140 was compared with RANTES, a natural CCR5 ligand that can inhibit HIV-1 entry by receptor downregulation as well as receptor blockade. Despite their divergent mechanisms of action and binding epitopes on CCR5, low nanomolar concentrations of both PRO 140 and RANTES inhibited infection of primary peripheral blood mononuclear cells (PBMC) by all CCR5-using (R5) viruses tested. This is consistent with there being a highly restricted pattern of CCR5 usage by R5 viruses. In addition, a panel of 25 subtype C South African R5 viruses were broadly inhibited by PRO 140, RANTES, and TAK-779, although ϳ30-fold-higher concentrations of the last compound were required. Interestingly, significant inhibition of a dualtropic subtype C virus was also observed. Whereas PRO 140 potently inhibited HIV-1 replication in both PBMC and primary macrophages, RANTES exhibited limited antiviral activity in macrophage cultures. Thus CCR5-targeting agents such as PRO 140 can demonstrate potent and genetic-subtype-independent anti-HIV-1 activity.

Resistance to small-molecule CCR5 inhibitors arises when HIV-1 variants acquire the ability to use inhibitor-bound CCR5 while still recognizing free CCR5. Two isolates, CC101.19 and D1/85.16, became resistant via four substitutions in the gp120 V3 region and three in the gp41 fusion peptide (FP), respectively. The binding characteristics of a panel of monoclonal antibodies (MAbs) imply that several antigenic forms of CCR5 are expressed at different levels on the surfaces of U87-CD4-CCR5 cells and primary CD4؉ T cells, in a cell-typedependent manner. CCR5 binding and HIV-1 infection inhibition experiments suggest that the two CCR5 inhibitor-resistant viruses altered their interactions with CCR5 in different ways. As a result, both mutants became generally more sensitive to inhibition by CCR5 MAbs, and the FP mutant is specifically sensitive to a MAb that stains discrete cell surface clusters of CCR5 that may correspond to lipid rafts. We conclude that some MAbs detect different antigenic forms of CCR5 and that inhibitor-sensitive and -resistant viruses can use these CCR5 forms differently for entry in the presence or absence of CCR5 inhibitors.The small-molecule CCR5 inhibitors maraviroc (MVC) and vicriviroc (VVC) are, or have been, used to treat human immunodeficiency virus type 1 (HIV-1) infection. They bind in the transmembrane helices and stabilize CCR5 in a conformation the viral Env complex cannot use efficiently (14,26,47). Resistant viruses usually gain the ability to enter cells via inhibitor-bound CCR5 while retaining the use of free CCR5 (46, 57). Virus-CCR5 binding involves interactions between the Tyr-sulfated N terminus (NT) and the second extracellular loop (ECL2) of the coreceptor and the 4-stranded bridging sheet and V3 region of the gp120 glycoprotein, respectively (20,21). In the most common genetic route to resistance, multiple sequence changes in V3 make the virus more dependent on the CCR5 NT (4,7,27,(37)(38)(39)55). A much rarer pathway involves changes in the fusion peptide (FP) of the gp41 protein, but the resistance mechanism is unknown (3). These pathways were followed when resistant isolates CC101.19 and D1/85.16 were derived from CC1/85 under selection by two similar inhibitors, AD101 and VVC, in peripheral blood mononuclear cells (PBMCs); the most critical resistance-associated substitutions in the escape mutant viruses were four in V3 and three in the FP (27,33). In this study, we used infectious Env chimeric clones, Res-4V3 derived from CC101.19 and Res-3FP from D1/85.16, together with the parental clones Par-4V3 and Par-3FP, derived from CC1/85, which were chosen based on sequence similarities with Res-4V3 and Res-3FP (7).The HIV-1 coreceptors CCR5 and CXCR4 exist in heterogeneous forms (6, 29), influenced by factors such as posttranslational modifications, coupling to G proteins, and the lipid environment (5,8,15,34,35). CCR5 monoclonal antibodies (MAbs) can vary considerably in how they stain different cell types in a way that is not always explained by CCR5 expression levels (1...

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The CC-chemokine receptor CCR5 is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of CD4+ cells and interacts directly with the viral surface glycoprotein gp120. Although receptor chimera studies have provided useful information, the domains of CCR5 that function for HIV-1 entry, including the site of gp120 interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of CCR5 to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish CCR5-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair Env-mediated membrane fusion and the gp120-CCR5 interaction. Of these three residues, only D11 is necessary for CC-chemokine-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites on CCR5 are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal CCR5 domain.

The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs’ abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events.