Characterization of gp120 and its Single-Chain Derivatives, $gp120-CD4_D_1_2$ and gp120-M9: Implications for Targeting the $CD4_i$ Epitope in Human Immunodeficiency Virus Vaccine Design

Varadarajan, Raghavan and Sharma, Deepak and Chakraborty, Kausik and Patel, Mayuri and Citron, Michael and Sinha, Prem and Yadav, Ramkishor and Rashid, Umar and Kennedy, Sarah and Eckert, Debra and Geleziunas, Romas and Bramhill, David and Schleif, William and Liang, Xiaoping and Shiver, John (2005) Characterization of gp120 and its Single-Chain Derivatives, $gp120-CD4_D_1_2$ and gp120-M9: Implications for Targeting the $CD4_i$ Epitope in Human Immunodeficiency Virus Vaccine Design. In: Journal of Virology, 79 (3). pp. 1713-1723.

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Abstract

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 $(gp120-CD4_D_1_2)$ or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that $gp120-CD4_D_1_2$ 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 $gp120-CD4_D_1_2$ to 17b is not affected by the addition of excess soluble $CD4_D_1_2$, 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 $CD4_D_1_2$. Immunogenicity studies of gp120, $gp120-CD4_D_1_2$, 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 $gp120-CD4_D_1_2$. These antisera were depleted of anti-$CD4_D_1_2$ antibodies by being passed over a column containing immobilized $CD4_D_1_2$. 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 $gp120-CD4_D_1_2$ 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 CD4i epitope were generated by the $gp120-CD4_D_1_2$ immunogen, these antibodies were nonneutralizing.

Item Type:	Journal Article
Publication:	Journal of Virology
Publisher:	American Society for Microbiology
Additional Information:	Copyright of this article belongs to American Society for Microbiology.
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	03 Mar 2006
Last Modified:	16 Jan 2013 11:00
URI:	http://eprints.iisc.ac.in/id/eprint/5689

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