A neutralizing recombinant human antibody Fab fragment against Puumala hantavirus.
A combinatorial human antibody Fab pComb3H library, generated from splenic lymphocytes of a Puumala hantavirus (PUUV) immune individual, was selected against PUUV using the phage display technique. Panning was carried out with antigens immobilized by MAbs directed to the two PUUV envelope glycoproteins G1 and G2. Thirteen Fabs, with reactivity directed to PUUV and specifically the G2 protein, as assessed by immunofluorescence and ELISA respectively, were isolated in crude preparations. By a focus reduction neutralization test (FRNT), four of the 13 crude Fab preparations exhibited type-specific neutralization of PUUV (strain Sotkamo) with 44-54% reduction in the number of foci. After affinity purification, the four Fab clones exhibited 50% focus reduction of PUUV at concentrations below 2 microg/ml. Sequencing of the heavy and light chain complementarity determining regions (CDR) 1-3 showed that the four selected clones were identical within the antibody binding regions. In inhibition tests with the PUUV G2-specific MAbs, 4G2 and 1C9, a new epitope important for neutralization, designated as G2-a3, was defined. This epitope, overlapping partially the neutralizing epitope recognized by the human MAb 1C9, seems to be unique for the PUUV serotype since none of the Fab clones neutralized any of the other hantaviruses tested.
de Carvalho Nicacio
C
C
Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden.
Lundkvist
A
A
Sjölander
K B
KB
Plyusnin
A
A
Salonen
E M
EM
Björling
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D006476:Orthohantavirus / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000276:immunology*
D000914:Antibodies, Viral; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C104008:glycoprotein G1, Hantavirus; C090197:glycoprotein G2, Hantavirus