EFFICIENT PURIFICATION AND RIGOROUS CHARACTERIZATION OF A RECOMBINANTGP120 FOR HIV VACCINE STUDIES

A recombinant HIV-1 gp120 (rgp120) was expressed in a permanent Chines e Hamster Ovary (CHO) cell line (L761h) that constitutively secretes t he product of clone p4 derived from the env gene of HIV-1 isolate GB8. The rgp120 was isolated from cell culture supernatants by a simple, r apid, non-denaturing and efficient purification procedure based on a n ovel combination of lectin affinity and FPLC ion-exchange chromatograp hy. The purity of the isolated glycoprotein was rigorously confirmed b y SDS-PAGE, capillary electrophoresis, laser desorption mass spectrome try, total amino acid analysis and N-terminal amino acid sequencing. T he retention of biological activity by the purified rgp120 was assesse d by determining the dissociation constant of rgp120 binding to sCD4. After formulation of this highly purified and biologically active rgp1 20 with ''conventional'' adjuvants, including types already used in cl inical trials of candidate gp120-based HIV vaccines, antibody response s in immunised rabbits were analysed using panels of overlapping synth etic peptides. The consequences of using currently available adjuvants to deliver highly specialised and perhaps conformation-dependent mole cules, like HIV gp120, are presented and discussed in the context of H IV vaccine development.