Efficient purification and rigorous characterisation of a recombinant gp120 for HIV vaccine studies.
A recombinant HIV-1 gp120 (rgp120) was expressed in a permanent Chinese Hamster Ovary (CHO) cell line (L761h) that constitutively secretes the 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, rapid, non-denaturing and efficient purification procedure based on a novel combination of lectin affinity and FPLC ion-exchange chromatography. The purity of the isolated glycoprotein was rigorously confirmed by SDS-PAGE, capillary electrophoresis, laser desorption mass spectrometry, total amino acid analysis and N-terminal amino acid sequencing. The retention of biological activity by the purified rgp120 was assessed by determining the dissociation constant of rgp120 binding to sCD4. After formulation of this highly purified and biologically active rgp120 with "conventional" adjuvants, including types already used in clinical trials of candidate gp120-based HIV vaccines, antibody responses in immunised rabbits were analysed using panels of overlapping synthetic peptides. The consequences of using currently available adjuvants to deliver highly specialised and perhaps conformation-dependent molecules, like HIV gp120, are presented and discussed in the context of HIV vaccine development.
Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK.
D H
DH
Jones
B W
BW
McBride
M A
MA
Roff
G H
GH
Farrar
D016915:AIDS Vaccines / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000737:chemistry; D016466:CHO Cells; D006224:Cricetinae; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D015483:HIV Antibodies / Q000096:biosynthesis / Q000737:chemistry; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000276:immunology
D016915:AIDS Vaccines; D015704:CD4 Antigens; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D014614:Vaccines, Synthetic