Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS

Abstract

The SARS-CoV spike glycoprotein (S) is the main target of the protective immune response in humans and animal models of SARS. Here, we demonstrated that efficient expression of S from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral RNA export elements, such as the CTE of Mazon-Pfizer monkey virus or the PRE of Woodchuck hepatitis virus (WPRE). The presence of both splice sites and WPRE markedly improved the immunogenicity of S-based DNA vaccines against SARS. Upon immunization of mice with low doses (2 microg) of naked DNA, only intron and WPRE-containing vectors could induce neutralizing anti-S antibodies and provide protection against challenge with SARS-CoV. Our observations are likely to be useful for the construction of plasmid and viral vectors designed for optimal expression of intronless genes derived from cytoplasmic RNA viruses.

Fig. 1

Transient expression of the SARS-CoV spike protein following plasmid DNA transfection. Subconfluent monolayers of Vero E6 cells were transfected with empty (−) or S (+) expression plasmids. At 48 h post-transfection, whole cell extracts were prepared in Laemmli sample buffer, separated by SDS-PAGE as indicated (lanes 1 to 4) and analyzed by Western blot as described in Materials and methods using rabbit polyclonal antibodies directed against the S protein. Alternatively, VeroE6 cells were infected with VVTF7-3 at a MOI of 5, transfected 1 h later with the indicated construct and analyzed for S expression 18 h thereafter (lanes 5 and 6). As controls, whole cell extracts prepared from VeroE6 cells either mock infected (lane 7) or infected with SARS-CoV (lane 8) were analyzed. The position of the molecular weight markers is shown on the right of the gel (kDa).

Fig. 2

Improvement of SARS-CoV spike protein expression by viral export elements. (A) Schematic representation of S expression plasmids. Viral export elements were inserted in pcDNA-S and pCI-S plasmids, downstream of the S ORF sequence and upstream of the polyadenylation signal (pA). SP: signal peptide. TM: transmembrane domain. SD-SA: donor and acceptor splice sites, respectively. BGH: bovine growth hormone. SV40: Simian Virus 40. WPRE: Woodchuck hepatitis virus post-transcriptional element. CTE: constitutive transport element from Mason Pfizer Monkey Virus. Subconfluent monolayers of VeroE6 (B) and 293T (C) cells were transfected with the indicated S expression constructs. At 48 h post-transfection, whole cell extracts were analyzed by Western blotting as in Fig. 1. The position of the molecular weight markers is shown on the right of the gel (kDa).

Fig. 3

Analysis of S protein expression at the surface of transfected cells. Subconfluent monolayers of 293T cells were transfected with each of the indicated S expression plasmids. At 48 h post-transfection, cells were detached from the plates and labeled with anti-S mouse polyclonal antibodies and FITC-conjugated anti-mouse IgG antibodies. Flow cytometry analysis was performed on a FACScalibur fluorocytometer. pCI-transfected 293T cells were used as control (dotted lines). S expression at the cell surface of transfected cells was quantified as the mean fluorescence intensity of the fraction of S-expressing cells (bar) and is indicated in each histogram in arbitrary units (1 = mean intensity measured in pCI-S-transfected cells).

Fig. 4

Analysis of S mRNA in transfected cells. Subconfluent monolayers of 293T cells were transfected with each of the indicated S expression plasmids. At 48 h post-transfection, cytoplasmic RNA was prepared and analysed by Northern blot for the presence of S mRNAs with a mixture of negative sense riboprobes specific for S sequences, as described in Materials and methods. The position of molecular weight markers is indicated on the side. For normalization, Northern blot analysis was performed in parallel using a negative sense β-actin specific riboprobe. The amounts of S specific RNAs of the expected size (stars) and β-actin RNAs were quantified and the calculated S/β-actin specific mRNA ratios are indicated below the images (1 = ratio found in pCI-S transfected cells).

Fig. 5

Efficient fusion of S-protein- and ACE2-expressing 293T cells. 293T cells constitutively expressing GFP (effector cells), were transfected with pCI-S-WPRE or, as control, with the empty pCI plasmid. 293T cells transfected with pCMV-DsRed (target cells) were also transfected with either pcDNA-ACE2 or, as control, with the empty pcDNA plasmid. At 36 h post-transfection, target and effector cells were mixed at a 1:1 ratio and analysed by flow cytometry for GFP and DsRed expression 12 h thereafter. The indicated percentage of fusion was measured as the ratio between double-stained cells (upper right quadrant) and red target cells (lower plus upper right quadrant). A: Effector cells transfected with pCI mixed with target cells transfected with pcDNA. B: Effector cells transfected with pCI-S-WPRE mixed with target cells transfected with pcDNA. C: Effector cells transfected with pCI mixed with target cells transfected with pcDNA-ACE2. D: Effector cells transfected with pCI-S-WPRE mixed with target cells transfected with pcDNA-ACE2.

Fig. 6

Serological evaluation of convalescent human sera using S-protein-expressing VeroE6 cells. Subconfluent monolayers of VeroE6 cells were transfected with pCI (A, B) or pCI-S-WPRE (C to F). At 48 h post-transfection, cells were stained as described in Materials and methods using either serum from patient A (#033168; B, E), or serum collected from patient B 8 days (#032703; C) or 29 days (#033153; F) after the onset of SARS symptoms, or serum collected before the SARS epidemic from a healthy donor (TV262; A, D).

Fig. 7

WPRE enhances anti-S antibody responses in mice immunized with low doses of plasmid DNA. Groups of 6 BALB/c mice were injected twice intramuscularly at 4-week interval with 2, 10 or 50 μg of S expression plasmid DNA as indicated, or with 50 μg of pCI plasmid DNA as control. Immune sera were collected 3 weeks after the second injection. SARS-CoV specific IgG antibody titers were determined by indirect ELISA using SARS-CoV infected VeroE6 cell lysates as the capture antigen, as described in Materials and methods. Values for each individual mouse are represented with black circles, and means with horizontal bars. The detection limit of the assay is indicated by a dotted line.

Fig. 8

WPRE is required for protection of mice from SARS-CoV challenge upon immunization with low-dose plasmid DNA. Groups of 8 BALB/c mice were injected three times intramuscularly at 4-week interval with 2 μg of S expression plasmid DNA as indicated, or with 2 μg of pCI plasmid DNA as control. Immune sera were collected 3 weeks after the third injection and assayed for neutralizing antibodies against SARS-CoV. Neutralization titers (A) were calculated as the reciprocal of the highest dilution of each individual serum, which completely prevented SARS-CoV CPE in 50% of the wells, as described in Materials and methods. Eight weeks after the third DNA injection, mice were challenged intranasally with SARS-CoV (10⁵ pfu/mouse). Two days after inoculation, mice were euthanized. Lung homogenates were prepared and titrated for infectious SARS-CoV by plaque assay on VeroE6 cells. Viral titers were expressed as log₁₀ (pfu/lungs) for individual mice (B). Values for each individual mouse are represented with black circles, and means with horizontal bars. The detection limits of the assays are indicated by a dotted line. Data shown are from one experiment representative of two.