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Journal of Virology, April 2008, p. 4125-4134, Vol. 82, No. 8
0022-538X/08/$08.00+0     doi:10.1128/JVI.02232-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Claudia Pastori,¹ Alberto Clivio,² Lorenzo Diomede,¹ Roberto Consonni,³ Giacomo M. S. De Mori,⁴ Renato Longhi,⁴ Giorgio Colombo,⁴ and Lucia Lopalco^1*

Infectious Diseases Clinic, San Raffaele Scientific Institute, Milan, Italy,¹ Università degli Studi di Milano, Milan, Italy,² Istituto per lo Studio delle Macromolecole, CNR, Milan, Italy,³ Istituto di Chimica del Riconoscimento molecolare, CNR, Milan, Italy⁴

Received 15 October 2007/ Accepted 28 January 2008

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 (A₉₅-A₉₆) increased antibody-peptide binding compared to that of the wild-type peptide (Asp₉₅-Phe₉₆). 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 A₉₅-A₉₆ 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.

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* Corresponding author. Mailing address: Infectious Diseases Clinic, San Raffaele Scientific Institute, Milan, Italy. Phone: 39-02-2643-7936. Fax: 39-02-2643-5381. E-mail: lopalco.lucia{at}hsr.it

Published ahead of print on 6 February 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, April 2008, p. 4125-4134, Vol. 82, No. 8
0022-538X/08/$08.00+0     doi:10.1128/JVI.02232-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.