Longitudinally Profiling Neutralizing Antibody Response to SARS Coronavirus with Pseudotypes

Nigel J. Temperton; Paul K. Chan; Graham Simmons; Maria C. Zambon; Richard S. Tedder; Yasuhiro Takeuchi; Robin A. Weiss


Emerging Infectious Diseases. 2005;11(3):411-416. 

In This Article

Materials and Methods

A total of 166 blood samples were obtained from 41 patients (68% female) 11-80 years of age who were admitted to the Prince of Wales Hospital, Hong Kong, from March to May 2003. All study patients fulfilled the World Health Organization criteria for having a probable case of SARS. Samples from 7 of the 41 patients were tested for SARS-CoV by reverse transcription-polymerase chain reaction (RT-PCR) in a study previously described,[31] and 4 patients had positive results. Pneumonia developed in all 41 patients, and 6 required intensive care. None of these patients died of the infection. For most patients, multiple samples were obtained at sequential times covering the acute, convalescent, and recovered phase of the disease. This study was approved by the Prince of Wales Hospital local institutional ethics committee.

Construction of the plasmid pCAGGS-S harboring full-length SARS-CoV S from the Urbani strain has been described previously.[23] The MLV gag/pol construct, pCMVi, and the green fluorescent protein (GFP) reporter construct, pCNCG, have been described.[32] Vesicular stomatitis virus envelope protein (VSV-G) expression vector pMDG has been described previously.[33] HIV constructs were used as described.[34]

All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) with Glutamax and high glucose (Gibco, Paisley, Scotland, UK), supplemented with 10% fetal calf serum and penicillin/streptomycin. To make the quail QT6/ACE2 cell line, the gene encoding the receptor for SARS-CoV, human angiotensin-converting enzyme 2 (ACE2),[35] was cloned from a human primary kidney cDNA library (Invitrogen, Paisley, Scotland, UK) using 21-mer primers designed to the start and stop of ACE2, and subcloned into pcdna3.1+. QT6 cells were transfected by using lipofectamine 2000 and selected with G418, and a bulk ACE2-positive, G418-resistant population was grown.

Confluent plates of 293T cells were split 1:4 the day before transfection. Each plate of 293T cells was transfected with 1 µg gag/pol construct, 1.5 µg of enhanced GFP reporter construct, and 1.5 µg envelope-expressing construct by using the Fugene-6 transfection reagent.[36] Supernatant was harvested 48 h and 72 h posttransfection, filtered through 0.45-µm filters, and stored at -80°C. MLV and HIV vector titer were measured on 293T, TE671, and QT6/ACE2 cells and are presented as infectious units (IU) per milliliter. Briefly, cells were infected with vector, and eGFP titers were determined 72 h later by fluorescence-activated cell sorter (FACS).

Live Virus. Patient serum samples were heat inactivated at 56°C for 30 min and serially diluted from 1:10 in culture medium. Fifty PFU of SARS Frankfurt strain were added to the serum dilution and incubated for 1 h at 37°C. We added 5 × 104 Vero E6 cells per well to the virus and serum mix, and the mixture was incubated in 96-well plates for 4 days, after which neutralization was assessed by cytopathic effect (CPE). The neutralization endpoint was taken as the last well in which complete neutralization was observed. Serum samples were assayed in duplicate, and positive results were confirmed in separate assays.

Pseudotype. Patient serum samples were heat inactivated at 56°C for 30 min, 2-fold serially diluted from 1:10 in culture medium, and mixed with MLV(SARS) virions (≈100 IU) at a 1:1 vol/vol ratio. After incubation at 37°C for 1 h, 100 µL of each dilution was added to QT6/ACE2 cells seeded at 1 × 104 cells per well in 96-well flat-bottomed tissue culture plates seeded 24 h previously. GFP-positive cells were counted 48 h later by fluorescence microscopy. Neutralizing antibody titers are presented as geometric mean titers of assays performed in triplicate.


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