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Journal of Virology, October 2003, p. 10984-10993, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.10984-10993.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Monoclonal Antibody 667 Recognizes the Variable Region A Motif of the Ecotropic Retrovirus CasBrE Envelope Glycoprotein and Inhibits Env Binding to the Viral Receptor

Hanna Dreja,1 Laurent Gros,1 Sylvie Villard,2 Estanislao Bachrach,1,{dagger} Anna Oates,1,{ddagger} Claude Granier,2 Thierry Chardes,3 Jean-Claude Mani,2 Marc Piechaczyk,1 and Mireia Pelegrin1*

Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, IFR 122, 34293 Montpellier Cédex 5,1 CNRS UMR 5094, Faculté de Pharmacie, 34093 Montpellier Cédex 5,2 CNRS UMR 5087, 30380 Saint Chrisol-lez-Alès, France3

Received 14 February 2002/ Accepted 7 July 2003

Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D57, is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

* Corresponding author. Mailing address: Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, IFR 122, 1919 Route de Mende, 34293 Montpellier Cédex 5, France. Phone: (33) 4 67 61 36 68. Fax: (33) 4 67 04 02 31. E-mail: pelegrin{at}igm.cnrs-mop.fr.

{dagger} Present address: Children's Hospital, Genetics Department, Boston, MA 02215.

{ddagger} Present address: MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

Journal of Virology, October 2003, p. 10984-10993, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.10984-10993.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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