Human Neutralizing Fab Molecules against Severe Acute Respiratory Syndrome Coronavirus Generated by Phage Display

Abstract

Human recombinant Fab fragments specific for the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV) were screened from a human Fab library, which was generated from RNAs from peripheral lymphocytes of convalescent SARS patients. Among 50 randomly picked clones, 12 Fabs specially reacted with S protein by an enzyme-linked immunosorbent assay. The microneutralizing test showed that one clone, designated M1A, had neutralizing activity on Vero E6 cells against SARS-CoV. DNA sequence analysis indicated that the light-and heavy-chain genes of M1A Fab belong to the 2a and 4f families, respectively. A neutralizing test on purified M1A demonstrated that 0.5 mg/ml of M1A completely inhibited SARS-CoV activity, with an absence of cytopathic effect for 7 days. Real-time fluorescence reverse transcription-PCR also proved the neutralizing capacity of M1A. These data showed that the number of virus copies was significantly reduced in the M1A-treated group, suggesting an important role for M1A in passive immunoprophylaxis against the SARS virus.During 2002 to 2003, severe acute respiratory syndrome (SARS) broke out worldwide. There is no effective medicine to cure this disease. Previous studies showed SARS patients could make great progress if they were given serum from convalescent SARS patients, indicating that generation of a human monoclonal antibody (MAb) against SARS coronavirus (SARS-CoV) may be helpful for passive immunoprophylaxis against SARS virus (8). The spike protein (S protein) of SARS-CoV is an important target for neutralizing antibody (9). We constructed a human antibody library by the phage display technique and used the S protein of SARS-CoV as the target to screen the phage antibody library. This study describes the generation of one neutralizing SARS-CoV-specific human antibody Fab molecule by panning selection of combinatorial Fab libraries against S proteins and quantitative analysis of the binding and neutralizing activity of the Fab molecules. MATERIALS AND METHODSConstruction of a Fab phage display library. Total RNA was extracted from peripheral blood lymphocytes of convalescent SARS patients (RNA isolation kit; Stratagene), and mRNA was reverse transcribed into cDNA using an oligo(dT) primer (Gibco/BRL). Fd segment (variable and first constant domains) genes and light-chain genes were amplified by using primers specific for the human chain genes (1). Then the amplified chains were cloned into the pComb3 phage display vector as described by Bjorling et al. (4). The ligated products were transformed into Escherichia coli XL1-Blue and resulted in a library of 2 ϫ 10 6 clones. The transformants were expanded into a volume of 2 liters, and the resulting phage was recovered as described previously (4).Enrichment of antigen-binding clones by panning. Microtiter wells were coated overnight at 4°C with 50 l of purified SARS-CoV (Beijing 01 strain) lysate antigen (30 g/ml), and after a blocking step with 3% bovine serum albumin (BSA)-phosphate-buffered saline (PBS), 50 l Fab phages (10 ...

Keeping It Together: Structures, Functions, and Applications of Viral Decoration Proteins

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