Isolation and characterization of selective and potent human Fab inhibitors directed to the active-site region of the two-component NS2B-NS3 proteinase of West Nile virus.
There is a need to develop inhibitors of mosquito-borne flaviviruses, including WNV (West Nile virus). In the present paper, we describe a novel and efficient recombinant-antibody technology that led us to the isolation of inhibitory high-affinity human antibodies to the active-site region of a viral proteinase. As a proof-of-principal, we have successfully used this technology and the synthetic naive human combinatorial antibody library HuCAL GOLD(R) to isolate selective and potent function-blocking active-site-targeting antibodies to the two-component WNV NS (non-structural protein) 2B-NS3 serine proteinase, the only proteinase encoded by the flaviviral genome. First, we used the wild-type enzyme in antibody screens. Next, the positive antibody clones were counter-screened using an NS2B-NS3 mutant with a single mutation of the catalytically essential active-site histidine residue. The specificity of the antibodies to the active site was confirmed by substrate-cleavage reactions and also by using proteinase mutants with additional single amino-acid substitutions in the active-site region. The selected WNV antibodies did not recognize the structurally similar viral proteinases from Dengue virus type 2 and hepatitis C virus, and human serine proteinases. Because of their high selectivity and affinity, the identified human antibodies are attractive reagents for both further mutagenesis and structure-based optimization and, in addition, for studies of NS2B-NS3 activity. Conceptually, it is likely that the generic technology reported in the present paper will be useful for the generation of active-site-specific antibody probes for multiple enzymes.
Inflammatory and Infectious Disease Center, The Sanford-Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
Sergey A
SA
Shiryaev
Ilian A
IA
Radichev
Boris I
BI
Ratnikov
Alexander E
AE
Aleshin
Katarzyna
K
Gawlik
Boguslaw
B
Stec
Christian
C
Frisch
Achim
A
Knappik
Alex Y
AY
Strongin
D000914:Antibodies, Viral / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014902:West Nile virus / Q000201:enzymology* / Q000235:genetics
D000914:Antibodies, Viral; D017361:Viral Nonstructural Proteins; D057057:Serine Proteases