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Journal of Virology, February 2005, p. 1713-1723, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1713-1723.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of gp120 and Its Single-Chain Derivatives, gp120-CD4_D12 and gp120-M9: Implications for Targeting the CD4_i Epitope in Human Immunodeficiency Virus Vaccine Design

Raghavan Varadarajan,^1* Deepak Sharma,¹ Kausik Chakraborty,¹ Mayuri Patel,² Michael Citron,³ Prem Sinha,¹ Ramkishor Yadav,¹ Umar Rashid,¹ Sarah Kennedy,² Debra Eckert,² Romas Geleziunas,³ David Bramhill,² William Schleif,³ Xiaoping Liang,³ and John Shiver³

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India,¹ Merck Research Labs, Rahway, New Jersey,² Merck Research Labs, West Point, Pennsylvania³

Received 20 June 2004/ Accepted 20 September 2004

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4_D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4_D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4_D12 to 17b is not affected by the addition of excess soluble CD4_D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4_D12. Immunogenicity studies of gp120, gp120-CD4_D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4_D12. These antisera were depleted of anti-CD4_D12 antibodies by being passed over a column containing immobilized CD4_D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4_D12 competed with the CD4_i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4_i epitope were generated by the gp120-CD4_D12 immunogen, these antibodies were nonneutralizing.

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* Corresponding author. Mailing address: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India. Phone: (91) 80-22932612. Fax: (91) 80-23600535. E-mail: varadar{at}mbu.iisc.ernet.in.

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Journal of Virology, February 2005, p. 1713-1723, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1713-1723.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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