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

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