title: Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design.
abstract: Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 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-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, 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 CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, 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-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. 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-CD4D12 competed with the CD4i 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-CD4D12 immunogen, these antibodies were nonneutralizing.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India. varadar@mbu.iisc.ernet.in
authors.forename: Raghavan
authors.initials: R
authors.lastname: Varadarajan
authors.forename: Deepak
authors.initials: D
authors.lastname: Sharma
authors.forename: Kausik
authors.initials: K
authors.lastname: Chakraborty
authors.forename: Mayuri
authors.initials: M
authors.lastname: Patel
authors.forename: Michael
authors.initials: M
authors.lastname: Citron
authors.forename: Prem
authors.initials: P
authors.lastname: Sinha
authors.forename: Ramkishor
authors.initials: R
authors.lastname: Yadav
authors.forename: Umar
authors.initials: U
authors.lastname: Rashid
authors.forename: Sarah
authors.initials: S
authors.lastname: Kennedy
authors.forename: Debra
authors.initials: D
authors.lastname: Eckert
authors.forename: Romas
authors.initials: R
authors.lastname: Geleziunas
authors.forename: David
authors.initials: D
authors.lastname: Bramhill
authors.forename: William
authors.initials: W
authors.lastname: Schleif
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Liang
authors.forename: John
authors.initials: J
authors.lastname: Shiver
mesh_terms: D016915:AIDS Vaccines / Q000008:administration & dosage / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D015704:CD4 Antigens / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002942:Circular Dichroism; D015195:Drug Design; D000939:Epitopes; D015483:HIV Antibodies / Q000276:immunology / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D009500:Neutralization Tests; D020349:Surface Plasmon Resonance
chemical_list: D016915:AIDS Vaccines; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120