Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): identification of neutralizing and antibodies reactive to S, N, M and E viral proteins

Abstract

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Fig. 1

Immunofluorescence staining (IFA) of SARS-CoV infected Vero E6 cells. A representative IFA is shown for an S protein-specific mAb (mAb 341) and M protein-specific mAb (mAb 292). Cell staining was visualized at 40× and 100× using a Zeiss Axioskop microscope with an Axiovert BlueH 485 nm filter and an RT Color Spot digital camera.

Fig. 2

Radioimmunoprecipitation of mAbs with SARS-CoV lysate. SARS-CoV-infected Vero E6 cells (+) or mock-infected Vero E6 cells (−) were radiolabeled with [³⁵S]-methionine/cysteine and reacted with individual mAbs to determine antigen specificity as described previously in Section 2. The anti-SARS-CoV S (S) mAbs (341, 534, 560, and 240) detected a protein with an apparent molecular mass of approximately 150 kDa, while the anti-matrix (M, 283) and anti-nucleocapsid (N, 42) mAbs detected proteins with an apparent molecular mass of approximately 25 kDa and 45 kDa, respectively. No bands were detected in the uninfected Vero E6 lysate control. The lack of signal with mAb 154 is likely due to the differential binding properties of protein G.

Fig. 3

Schematic diagram of the various S glycoprotein fragments synthesized. The top box represents the whole length of SARS-CoV S glycoprotein (aa 1–1255) and the relative sizes of the selected S protein fragments are shown.

Fig. 4

Epitope mapping of neutralizing and non-neutralizing anti-S mAbs. The epitopes recognized by the anti-S protein mAbs were determined by Western Blot with C-terminally truncated S proteins covering the region between amino acids 269 and 1190 of the S protein. S1190, aa 1–1190; S510, aa 1–510; S490, aa 1–490; S350, aa 1–350; S270–510, aa 270–510; S269, aa 1–269. A representative Western Blot for a neutralizing anti-S protein monoclonal, 341C (A), a non-neutralizing anti-S protein monoclonal, 560C (B), SARS convalescent-phase sera (C) and anti-(His)₆ antibody (D) are shown.