Selection of Active ScFv to G-Protein-Coupled Receptor CCR5 Using Surface Antigen-Mimicking Peptides

Zhang, Ying; Pool, Chadler T.; Sadler, Kristen; Hong, Yan; Edl, Jennifer; Wang, Xiaohong; Boyd, James G.; Tam, James P.

doi:10.1021/bi0492152

Cited by 31 publications

(27 citation statements)

References 37 publications

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“…Having shown that the linear synthetic peptide CCR5 89-102 is poorly immunogenic, we turned our attention to its cyclic form and to the FHV Epitope Presentation System. Analogous to our results, a recent report has shown that it is possible to select anti-CCR5 single-chain variable fragments from a phage display antibody library and that only those selected by binding to cyclic constrained peptides inhibited CCR5-mediated (but not CXCR4-mediated) HIV infection (40).…”

Section: Discussionsupporting

confidence: 87%

Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

Barassi¹,

Soprana²,

Pastori³

et al. 2005

J Virol

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The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4 ؉ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4؉ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4 ؉ cells from both humans and untreated mice, (iii) inhibit Mip-1␤ chemotaxis of CD4 ؉ CCR5 ؉ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

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Section: Discussionsupporting

confidence: 87%

Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

Barassi¹,

Soprana²,

Pastori³

et al. 2005

J Virol

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“…Cyclic peptides have also been successful in the isolation of scFv phage antibodies targeted to the extracellular loops of CCR5. 53 Chemical Linkage of Peptides onto Scaffolds (CLiPS) technology has also been used successful to generate functional antibodies to CXCR7.…”

Section: Immunogen Formats Used For Gpcrsmentioning

confidence: 99%

Therapeutic antibodies directed at G protein-coupled receptors

Hutchings

Koglin²,

Marshall³

2010

mAbs

148

130

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“…In addition, the second loop is mostly responsible for the binding of the endogenous chemokine peptides (42). Moreover, very recently a high level of immunogenicity has been found to both the N terminus and the first cysteine loop (49). Taken together, these findings suggest that the external domains of CCR5 are not clearly independent.…”

mentioning

confidence: 99%

Two Amino Acid Substitutions within the First External Loop of CCR5 Induce Human Immunodeficiency Virus-Blocking Antibodies in Mice and Chickens

Pastori¹,

Clivio

Diomede³

et al. 2008

J Virol

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Antibodies to the first loop (ECL1) of CCR5 have been identified in human immunodeficiency virus (HIV)-exposed uninfected individuals (ESN) and in HIV-positive nonprogressing subjects. Thus, these antibodies may confer resistance against HIV infection. To define which amino acids are involved in antibody binding to CCR5, we performed a peptide-scanning assay and studied the immunogenicity of peptides in animal models. A panel of synthetic peptides spanning the CCR5-ECL1 region and displaying glycine or alanine substitutions was assayed for antibody binding with a pool of natural anti-CCR5 antibodies. We used mice and chickens to study the immunogenicity of mutagenized peptide. Structural characterization by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations were performed to better understand the structural and conformational features of the mutagenized peptide. Amino acid substitutions in positions Ala95 and Ala96 (A 95 -A 96 ) increased antibody-peptide binding compared to that of the wild-type peptide (Asp 95 -Phe 96 ). The Ala95-96 peptide was shown to induce, in mice and chickens, antibodies displaying biological activity at very low concentrations. Strikingly, chicken antibodies to the Ala95-96 peptide specifically recognize human CCR5 molecules, downregulate receptors from lymphocytes, inhibit CCR5-dependent chemotaxis, and prevent infection by several R5 viruses, displaying 50% inhibitory concentrations of less than 3 ng/ml. NMR spectroscopy and molecular dynamics simulations proved the high flexibility of isolated epitopes and suggested that A 95 -A 96 substitutions determine a slightly higher tendency to generate helical conformations combined with a lower steric hindrance of the side chains in the peptides. These findings may be relevant to the induction of strong and efficient HIV-blocking antibodies.The CCR5 coreceptor is a seven-transmembrane (TM)-spanning receptor involved in chemokine signaling (13,35,38). It also is used as a viral coreceptor by human immunodeficiency virus (HIV), and it presumably mediates the first contacts between HIV and target host cells in mucosal sites, as CCR5-tropic HIV strains are generally the first in pioneering new hosts (6,8). The HIV-gp120 glycoprotein binds preferentially to the N terminus of CCR5 and the second extracellular region (18, 20). In addition, the second loop is mostly responsible for the binding of the endogenous chemokine peptides (42). Moreover, very recently a high level of immunogenicity has been found to both the N terminus and the first cysteine loop (49). Taken together, these findings suggest that the external domains of CCR5 are not clearly independent. In fact, it has been shown that CCR5 possesses a very dynamic equilibrium in the course of its interaction with Env proteins, which may induce different conformations in vivo (36). The binding of anti-gp120 immunoglobulins (Igs) also is known to alter the local conformation of viral and cellular proteins involved in the complex and therefore affect the whole proc...

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Selection of Active ScFv to G-Protein-Coupled Receptor CCR5 Using Surface Antigen-Mimicking Peptides

Cited by 31 publications

References 37 publications

Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

Therapeutic antibodies directed at G protein-coupled receptors

Two Amino Acid Substitutions within the First External Loop of CCR5 Induce Human Immunodeficiency Virus-Blocking Antibodies in Mice and Chickens

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