CCR5 mAb Research Articles

Identifying CCR5 coreceptor populations permissive for HIV-1 entry and productive infection: implications for in vivo studies

BackgroundThe chemokine receptor CCR5 is the major coreceptor for HIV-1 cell entry. We previously observed that not all CCR5 mAbs reduce HIV-1 infection, suggesting that only some CCR5 populations are permissive for HIV-1 entry. This study aims to better understand the relevant conformational states of the cellular coreceptor, CCR5, involved in HIV entry. We hypothesized that CCR5 assumes multiple configurations during normal cycling on the plasma membrane, but only particular forms facilitate HIV-1 infection.MethodsTo this end, we quantified different CCR5 populations using six CCR5 monoclonal antibodies (mAbs) with different epitope specificities and visualized them with super-resolution microscopy. We quantified each surface CCR5 population before and after HIV-1 infection.ResultsBased on CCR5 conformational changes, down-modulation, and trafficking rates (internalization and recycling kinetics), we were able to distinguish among heterogeneous CCR5 populations and thus which populations might best be targeted to inhibit HIV-1 entry. We assume that a decreased surface presence of a particular CCR5 subpopulation following infection means that it has been internalized due to HIV-1 entry, and that it therefore represents a highly relevant target for future antiviral therapy strategies. Strikingly, this was most true for antibody CTC8, which targets the N-terminal region of CCR5 and blocks viral entry more efficiently than it blocks chemokine binding.ConclusionsDefining the virus-host interactions responsible for HIV-1 transmission, including specific coreceptor populations capable of establishing de novo infections, is essential for the development of an HIV-1 vaccine. This study hopefully will facilitate further development of inhibitors to block CCR5 usage by HIV-1, as well as inform future HIV-1 vaccine design.

Targeting inhibition of CCR5 on improving obesity-associated insulin resistance and impairment of pancreatic insulin secretion in high fat-fed rodent models

Obesity is closely linked with type 2 diabetes and the effective therapies on obesity-associated diabetes are under development. The aim of this study was undertaken to investigate whether the inhibition of the augmented CCR5-mediated signaling could be a common target for treatment of obesity-associated insulin resistance and impairment of pancreatic insulin secretion in high-fat diet (HFD) fed rats and CCR5 knockout mice and also in isolated islets and RIN-m5F cells. Conducted with SD rats, HFD-induced body weight gain was significantly decreased in those combined with Maraviroc treatment, but food intake remained similar compared to control. Maraviroc also significantly improved the impaired oral glucose tolerance test (OGTT). As compared with wild-type mice, CCR5 deletion significantly attenuated the HFD-induced increases in glucose area under curve of OGTT and the value of HOMA-IR as well as plasma lipid profile. It also reversed the HFD-suppressed gene expressions of GLUT4 and IRS-1 in adipose tissue. On the other hand, the HFD-associated islet macrophage and T-cell infiltration were significantly decreased in CCR5 KO mice. H2O2 significantly suppressed glucose-stimulated insulin secretion (GSIS) is isolated islets, which were significantly reversed in those cotreated with CCR5 mAb. H2O2 failed to change GSIS in those of CCR5 KO mice. The palmitate-induced reactive oxygen species production was significantly decreased in those cotreated with CCR5 antagonist in RIN-m5F cells. Collectively, it is suggested that targeting inhibition of the CCR5 mediated inflammatory pathway could not only improve obesity-associated insulin resistance but also directly alleviate pancreatic β-cell dysfunction.

2501. CCR5 Targeted ARV Loaded Nanoparticle: Dual Protection for HIV Functional Cure

BackgroundOne of the NIH high-priority HIV/AIDS research objectives is to discover novel therapeutics aimed at developing safe, tolerable, strategies that targets cellular protein to induce long-term antiviral suppression. This current research aims at designing a novel targeted nano-formulation combining cell targeting and antiretroviral therapy to provide double protection against HIV-1.MethodsCCR5 targeted combination antiretroviral drugs (cARV) loaded nanoparticles (NPs) were synthesized based on water-in-oil-in-water (W-O-W) emulsion methodology. For targeting CCR5+ T cells, a modified high affinity CCR5 monoclonal Ab (XFCCR5 mAb), was isolated from XF-CCR5 hybridoma cells. The XFCCR5 mAbs were covalently conjugated through their C-terminus by replacing the NHS group on FTC+DTG NPs with covalent amide bond. The CCR5 mAb binding was evaluated by SDS–PAGE methods. The CCR5-specific binding affinity of XFCCR5-FTC+DTG NP in compared with XFCCR5 monoclonal antibody (mAb) was evaluated by flow cytometry using CD4+CCR5+ TZM-bl cell line and PBMCs. The intracellular pharmacokinetic (PK) profile in TZMbl cells was evaluated by LC-MS/MS analysis, whereas in vitro efficacy was evaluated based on Steady-Glo® Luciferase Assay System using TZM-bl cells.ResultsXFCCR5-FTC+DTG NPs obtained averaged 173 ± 23 nm (mean ± SEM, n = 3) with 2.2 ± 0.47 mg XFCCR5 mAb bound per mg cARV NP. The formulation % entrapment efficiency of DTG and FTC respectively, to be 55 ± 1.6% and 42.6 ± 5.6%. The specific binding affinity (Km) of XFCCR5-FTC+DTG NP and XFCCR5 mAb were estimated to be 0.0057 and 0.0377, higher compared with wild-type anti-CCR5 mAb with higher Km value 0.303. Finally, the 4-day HIV-infection protection study result illustrates IC50 in case of XFCCR5-FTC+DTG NP and XFCCR5 NP to be as low as 0.0069 and 0.0031 µg/mL, respectively, compared with 1.771 µg/mL for XFCCR5 mAb (Figure 1, TZM-bl and Figure 2, PBMCs).ConclusionThis nano-formulation aimed at duel protection by preventing HIV binding to CCR5+CD4+T cell due to CCR5 receptor blocking. These result support protection against HIV and maintenance of cARV drug levels. This novel formulation could be supportive of immune-alternative to achieve functional-cure against HIV. Disclosures All authors: No reported disclosures.

CCR5 Conformations Are Dynamic and Modulated by Localization, Trafficking and G Protein Association

CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs) are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.

P1-014 - CCL5 and CCR5 Interaction Promotes Cell Motility in Human Osteosarcoma

ABSTRACT Background Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES) was originally recognized as a product of activated T cells and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown. Methods Multiple molecular and pharmacological approaches such as migration assay, flow cytometric analysis, quantitative real-time PCR, western blot analysis, transfection and reporter gene assay and establishment of stably transfected cells were used to determine CCL5-regulated effect and mechanism in human osteosarcoma cell lines (U2OS and MG63). Results We found that CCL5 increased the migration and expression of avb3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK and NF-kB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK and NF-kB cascades. In addition, overexpression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells. Conclusions These results suggest that the CCL5 and CCR5 interaction acts through the MEK/ERK signaling pathway, which in turn activates NF-kB, resulting in the activation of avb3 integrin and contributing the migration of human osteosarcoma cells.

CCL5 and CCR5 interaction promotes cell motility in human osteosarcoma.

BackgroundOsteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown.Methodology/Principal FindingsHere we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells.Conclusions/SignificanceCCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells.

Multiple CCR5 Conformations on the Cell Surface Are Used Differentially by Human Immunodeficiency Viruses Resistant or Sensitive to CCR5 Inhibitors

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-type-dependent 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.

Constrained use of CCR5 on CD4+ lymphocytes by R5X4 HIV-1: Efficiency of Env–CCR5 interactions and low CCR5 expression determine a range of restricted CCR5-mediated entry

R5X4 HIV-1 has impaired utilization of CCR5 on primary CD4+ lymphocytes but the mechanisms responsible are not well defined. Using a panel of diverse R5X4 Envs we identified a spectrum of CCR5 use on CD4+ lymphocytes. Greater lymphocyte CCR5 use correlated with relative resistance to CCR5 mAbs and small molecule antagonists. Increasing CCR5 expression on lymphocytes increased the proportion of entry mediated by CCR5 for all R5X4 isolates except 89.6. In cell lines with regulated CCR5 expression, strains with greater lymphocyte CCR5 use better exploited limiting levels of CCR5. Introduction of an R306S mutation in the 89.6 V3 domain enhanced its utilization of CCR5 at low levels and switched its preference to CCR5 for lymphocyte entry. Thus, the degree to which R5X4 HIV-1 use primary lymphocyte CCR5 is determined by low CCR5 expression coupled with variations in the efficiency of Env-CCR5 interactions, which is in part governed by V3 sequences.

CCR5 monoclonal antibodies for HIV-1 therapy

To summarize emerging clinical and preclinical data pertaining to the use of CCR5 monoclonal antibodies (mAbs) as therapies for HIV-1 infection. The epitope specificity of CCR5 mAbs is discussed in relation to its critical impact on antiviral activity and CCR5 antagonism. We compare and contrast mAbs and small-molecule CCR5 antagonists in terms of their binding and antiviral properties. Two CCR5 mAbs have entered clinical testing and have successfully completed proof-of-concept studies in HIV-infected individuals, providing initial information on the potential therapeutic utility of these agents. New studies support the view that the most potent antiviral CCR5 mAbs recognize the second extracellular loop of CCR5 either exclusively or in combination with the amino terminus. Studies have revealed fundamental differences in how mAbs and small molecules bind CCR5 and inhibit HIV-1. CCR5 mAbs and small-molecule CCR5 antagonists have demonstrated consistent antiviral synergy and limited or no viral cross-resistance in independent studies. Single intravenous infusions of CCR5 mAbs significantly reduced HIV-1 RNA levels in infected individuals for 2-3 weeks without appreciable toxicity. CCR5 mAbs have demonstrated broad and potent antiviral activity in vitro. Clinical studies have established CCR5 mAbs as potent antiretroviral agents with prolonged activity following a single dose. CCR5 mAbs represent both a distinct class of CCR5 inhibitor and a novel approach to HIV-1 therapy.

CCR5 Small-Molecule Antagonists and Monoclonal Antibodies Exert Potent Synergistic Antiviral Effects by Cobinding to the Receptor

A panel of four CCR5 monoclonal antibodies (mAbs) recognizing different epitopes on CCR5 was examined in CCR5-mediated cell-cell fusion assay, alone or in combination with a variety of small molecule CCR5 antagonists. Although no antagonism was observed between any of the CCR5 inhibitors, surprisingly potent synergy was observed between CCR5 mAbs and antagonists, and the synergistic activity was confirmed in other antiviral assays. Strong synergy was also observed between CCR5 inhibitors and the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide. There was no synergy observed between small molecule CCR5 inhibitors; however, potent synergy was observed between mAbs recognizing different parts of CCR5. In all synergistic combinations, greater synergy was achieved at higher percent inhibition levels. A negative correlation was found between the degree of synergy between the two classes of CCR5 inhibitors and the ability to compete each other for binding to the receptor. For example, the greatest synergy, observed between the mAb ROAb13 and the small molecule inhibitor maraviroc, did not interfere with binding to CCR5 for either inhibitor, whereas no synergy was found between mAb 45523 and maraviroc, which do compete for binding to CCR5. In addition, in contrast to a recent report, the CCR5 inhibitors tested here were found to inhibit the same stage of HIV entry. Based on the data presented here, we hypothesize that CCR5 inhibitors exert synergistic antiviral actions through a cobinding mechanism.

Initial Events in Establishing Vaginal Entry and Infection by Human Immunodeficiency Virus Type-1

SummaryUnderstanding the initial events in the establishment of vaginal human immunodeficiency virus type-1 (HIV-1) entry and infection has been hampered by the lack of appropriate experimental models. Here, we show in an ex vivo human organ culture system that upon contact in situ, HIV-1 rapidly penetrated both intraepithelial vaginal Langerhans and CD4+ T cells. HIV-1 entered CD4+ T cells almost exclusively by CD4 and CCR5 receptor-mediated direct fusion, without requiring passage from Langerhans cells, and overt productive infection ensued. By contrast, HIV-1 entered CD1a+ Langerhans cells primarily by endocytosis, by means of multiple receptors, and virions persisted intact within the cytoplasm for several days. Our findings shed light on the very earliest steps of mucosal HIV infection in vivo and may guide the design of effective strategies to block local transmission and prevent HIV-1 spread.

The Second Extracellular Loop of CCR5 Contains the Dominant Epitopes for Highly Potent Anti-Human Immunodeficiency Virus Monoclonal Antibodies

Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2.

Novel CCR5 monoclonal antibodies with potent and broad-spectrum anti-HIV activities

To identify monoclonal antibodies (mAbs) with high potency and novel recognition sites, more than 25,000 of mouse hybridomas were screened and 4 novel anti-human CCR5 mAbs ROAb12, ROAb13, ROAb14, and ROAb18 showing potent activity in cell–cell fusion (CCF) assay were identified. These mAbs demonstrated potent antiviral activities in both single-cycle HIV infection (IC 50 range: 0.16–4.3 μg/ml) and PBMC viral replication (IC 50 range: 0.02–0.04 μg/ml) assays. These potent antiviral effects were donor-independent. All 4 mAbs were also highly potent in the PhenoSense assay against 29 HIV isolates covering clade A through G. In all antiviral assays, these mAbs showed potency superior to the previously reported mAb 2D7 in side-by-side comparison studies. All 4 mAbs were also fully active against viruses resistant to HIV fusion inhibitor enfuvirtide and CCR5 antagonist maraviroc. Although ROAb12, ROAb14, and ROAb18 inhibited RANTES, MIP1α and MIP1β binding and cell activation, the other novel mAb ROAb13 was inactive in inhibiting cell activation by these three ligands. Furthermore, highly synergistic antiviral effects were found between mAb ROAb13 and 2D7 or ROAb12. In addition, none of these mAbs showed agonist activity or caused internalization of the CCR5 receptor.

Monoclonal antibodies and small-molecules as distinct subclasses of CCR5-targeted therapies for HIV-1

The chemokine receptor CCR5 provides a portal of entry for HIV-1 and serves a target for potential new antiretroviral therapies. CCR5 monoclonal antibodies (mAb) and small-molecule CCR5 antagonists potently block HIV-1 entry in vitro, and controlled clinical trials have provided initial proof-of-concept for this mode of therapy. PRO 140 is a humanized CCR5 mAb that broadly and potently blocks CCR5-mediated HIV-1 entry in preclinical settings, and this product has entered phase 1b testing in HIV-infected individuals. In this study, PRO 140 and small-molecule CCR5 antagonists in development were compared in terms of patterns of viral resistance, synergistic interactions, competition for CCR5 binding, and related properties. In studies that examined forced viral resistance to CCR5 inhibitors in vitro, we observed limited or no viral cross-resistance between PRO 140 and small-molecule CCR5 antagonists. Potent antiviral synergy was observed between PRO 140 and small-molecule CCR5 antagonists in vitro. In contrast, modest synergy or additive effects were observed between PRO 140 and enfuvirtide and between different small-molecule CCR5 antagonists used in combination. Synergy between PRO 140 and small-molecule CCR5 antagonists was explained in part by their non-reciprocal patterns ofA competition for CCR5 binding. Additional studies indicate that PRO 140 may act as a competitive CCR5 inhibitor while the small-molecule CCR5 antagonists act via allosteric mechanisms. Collectively, the findings suggest that mAb and small-molecule CCR5 drugs may provide complementary HIV-1 treatment subclasses.

Potent Antiviral Synergy between Monoclonal Antibody and Small-Molecule CCR5 Inhibitors of Human Immunodeficiency Virus Type 1

The chemokine receptor CCR5 provides a portal of entry for human immunodeficiency virus type 1 (HIV-1) into susceptible CD4(+) cells. Both monoclonal antibody (MAb) and small-molecule CCR5 inhibitors have entered human clinical testing, but little is known regarding their potential interactions. We evaluated the interactions between CCR5 MAbs, small-molecule CCR5 antagonists, and inhibitors of HIV-1 gp120, gp41, and reverse transcriptase in vitro. Inhibition data were analyzed for cooperative effects using the combination index (CI) method and stringent statistical criteria. Potent, statistically significant antiviral synergy was observed between the CCR5 MAb PRO 140 and the small-molecule CCR5 antagonists maraviroc (UK-427,857), vicriviroc (SCH-D), and TAK-779. High-level synergy was observed consistently across various assay systems, HIV-1 envelopes, CCR5 target cells, and inhibition levels. CI values ranged from 0.18 to 0.64 and translated into in vitro dose reductions of up to 14-fold. Competition binding studies revealed nonreciprocal patterns of CCR5 binding by MAb and small-molecule CCR5 inhibitors, suggesting that synergy occurs at the level of receptor binding. In addition, both PRO 140 and maraviroc synergized with the chemokine RANTES, a natural ligand for CCR5; however, additive effects were observed for both small-molecule CCR5 antagonists and PRO 140 in combination with other classes of HIV-1 inhibitors. The findings provide a rationale for clinical exploration of MAb and small-molecule CCR5 inhibitors in novel dual-CCR5 regimens for HIV-1 therapy.

R5- and X4-HIV-1 use differentially the endometrial epithelial cells HEC-1A to ensure their own spread: Implication for mechanisms of sexual transmission

The mechanism of viral transmission across the mucosal barrier is poorly understood. Using the endometrial epithelium-derived cell line HEC-1A, we found that the cells are capable of sequestering large numbers of HIV-1 particles but are refractory to cell-free viral infection. The removal of heparan sulfate moieties of cell-surface proteoglycans (HSPG) from the apical pole of HEC-1A accounted for at least 60% of both R5- and X4-HIV-1 attachment, showing their important implication in viral attachment. HEC-1A cells also have the capacity to endocytose a weak proportion of the attached virus and pass it along to underlying cells. Fucose, N-acetylglucosamine and mannosylated-residues inhibited the transcytosis of some virus isolates, suggesting that mannose receptors can be implicated on the both R5- and X4-HIV-1 transcytosis. The inhibition of HIV transcytosis by blocking CCR5 mAb suggests the implication of specific interaction between the viral gp120 and sulfated moiety of syndecans during the transcytosis of mostly R5- and X4-HIV-1. At the basolateral pole of HEC-1A, HSPG sequestered X4- and not R5-HIV-1, highlighting the important role of HEC-1A as an X4 virus reservoir. The cell-free virus particles that have transcytosed could infect activated T cells but with a weaker efficiency than virus that had not transcytosed. The specific stimulation of HEC-1A by R5-HIV-1 increased the release of monocytes/chemokines-attracting chemokines (IL-8 and GR0) and proinflammatory cytokines (TNF-β and IL-1α) that enhanced the production of virus by activated T cells. This study suggests that R5 and X4 viruses can differentially use epithelial cells to ensure their own spread.