Cloning and biological characterization of human single-chain Fv fragments that mediate neutralization of HIV-1.
OBJECTIVE
To develop recombinant single-chain Fv fragments against HIV-1 gp120.


METHODS
A panel of human monoclonal antibody Fv fragments were generated against the HIV-1 gp120 by affinity selection from an antibody library expressed on the surface of filamentous phage. The library was prepared from peripheral blood lymphocytes of an asymptomatic HIV-1-infected mother with a high neutralization titer. This mother did not transmit HIV-1 to her offspring (non-transmitter). Heavy and light chains were initially amplified separately and combined by splicing by overlap extension to generate Fv fragments.


RESULTS
Several clones expressing single-chain Fv fragments bind strongly to HIV-1 gp120 and several were found to neutralize cell-free HIV-1IIIB. Gross epitope mapping suggest that different clones bound to different functional regions on the envelope. The clones also exhibited sequence diversity.


CONCLUSIONS
This strategy of cloning resulted in the development of functional human-derived antibody reagents with different anti-HIV-1 biological properties in vitro. These recombinant Fv fragments have potential utility as immune reagents, as well as in the design of potential immunotherapeutics. In addition, these antibody reagents may provide information on the relationship between humoral immunity and maternal-fetal (vertical) HIV-1 transmission.
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
V
V
Srikantan
B
B
Wang
M A
MA
Satre
K E
KE
Ugen
K
K
Dang
F
F
Scales
A P
AP
Godillot
W V
WV
Williams
D B
DB
Weiner
D000163:Acquired Immunodeficiency Syndrome / Q000517:prevention & control / Q000635:transmission*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015497:HIV-1; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000627:therapeutic use; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000627:therapeutic use; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis; D007231:Infant, Newborn; D018445:Infectious Disease Transmission, Vertical / Q000517:prevention & control*; D008214:Lymphocytes / Q000276:immunology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000276:immunology* / Q000821:virology; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000627:therapeutic use
D017931:DNA Primers; D015699:HIV Envelope Protein gp120; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; C055966:immunoglobulin Fv