Characterization and HIV-1 fusion inhibitory properties of monoclonal Fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of HIV-1 gp41.
Using a human non-immune phage library comprising more than 10(9) functional human antibody specificities in Fab format, we have been able to select a set of eight monoclonal Fabs targeted against diverse epitopes of the ectodomain of gp41 from HIV-1. The antigens used for panning the antibodies comprised two soluble, disulfide-linked, trimeric polypeptides derived from gp41, N(CCG)-gp41 and N35(CCG)-N13. The former comprises an exposed trimeric coiled-coil of the N-helices of gp41 fused in helical phase to the minimal thermostable ectodomain of gp41, while the latter comprises only the trimeric coiled-coil of N-helices. The selected Fabs were probed by Western blot analysis against four antigens: N(CCG)-gp41, N35CCG-N13, N34CCG (a smaller version of N35CCG-N13), and the minimal thermostable ectodomain core of gp41 in its six-helix bundle conformation (6-HB). Three classes of Fabs were found: class A (two Fabs) interact predominantly with the 6-HB; class B (four Fabs) interact with both the 6-HB and the internal trimeric coiled-coil of N-helices; and class C (two Fabs) interact specifically with the internal trimeric coiled-coil of N-helices. The IC50 values for the Fabs, expressed as bivalent mini-antibodies, ranged from 6 microg/ml to 60 microg/ml in a quantitative vaccinia virus-based reporter gene assay for HIV-1 envelope-mediated cell fusion using the envelope from the HIV-1 T tropic strain LAV. The two most potent fusion inhibitors belonged to class B. This panel of Fabs provides a set of useful probes for studying HIV-1 envelope-mediated cell fusion and may serve as a basis for developing Fab-based anti-HIV-1 therapeutics.
Louis
John M
JM
Laboratory of Chemical Physics National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room B1/30I, Bethesda, MD 20892, USA.
Bewley
Carole A
CA
Gustchina
Elena
E
Aniana
Annie
A
Clore
G Marius
GM
D019380:Anti-HIV Agents; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D000914:Antibodies, Viral / Q000494:pharmacology; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D015700:HIV Envelope Protein gp41 / Q000276:immunology*; D015497:HIV-1 / Q000187:drug effects* / Q000276:immunology / Q000502:physiology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000494:pharmacology*; D008561:Membrane Fusion / Q000187:drug effects*; D019151:Peptide Library; D017434:Protein Structure, Tertiary
D019380:Anti-HIV Agents; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D015700:HIV Envelope Protein gp41; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library