Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 101 (26), 9804-9

Contributions of the Structural Proteins of Severe Acute Respiratory Syndrome Coronavirus to Protective Immunity

Affiliations

Contributions of the Structural Proteins of Severe Acute Respiratory Syndrome Coronavirus to Protective Immunity

Ursula J Buchholz et al. Proc Natl Acad Sci U S A.

Abstract

We investigated the contributions of the structural proteins of severe acute respiratory syndrome (SARS) coronavirus (CoV) to protective immunity by expressing them individually and in combinations from a recombinant parainfluenza virus (PIV) type 3 vector called BHPIV3. This vector provided direct immunization of the respiratory tract, the major site of SARS transmission, replication, and disease. The BHPIV3/SARS recombinants were evaluated for immunogenicity and protective efficacy in hamsters, which support a high level of pulmonary SARS-CoV replication. A single intranasal administration of BHPIV3 expressing the SARS-CoV spike protein (S) induced a high titer of SARS-CoV-neutralizing serum antibodies, only 2-fold less than that induced by SARS-CoV infection. The expression of S with the two other putative virion envelope proteins, the matrix M and small envelope E proteins, did not augment the neutralizing antibody response. In absence of S, expression of M and E or the nucleocapsid protein N did not induce a detectable serum SARS-CoV-neutralizing antibody response. Immunization with BHPIV3 expressing S provided complete protection against SARS-CoV challenge in the lower respiratory tract and partial protection in the upper respiratory tract. This was augmented slightly by coexpression with M and E. Expression of M, E, or N in the absence of S did not confer detectable protection. These results identify S among the structural proteins as the only significant SARS-CoV neutralization antigen and protective antigen and show that a single mucosal immunization is highly protective in an experimental animal that supports efficient replication of SARS-CoV.

Figures

Fig. 1.
Fig. 1.
Maps of the genomes of BHPIV3 vectors expressing the SARS-CoV S, M, E, and N ORFs or a heterologous control sequence equivalent in length to the S ORF. The BPIV3 backbone genes are shown as open rectangles, the HPIV3 surface protein genes are checked rectangles, and the SARS-CoV ORFs and control sequence are shaded rectangles. PIV3 gene-start and gene-end transcription signals are shown as black triangles and bars, respectively, flanking each rectangle. The figure is not drawn to scale. le, leader; tr, trailer.
Fig. 2.
Fig. 2.
Transcription of the SARS-CoV inserts into monocistronic mRNAs. LLC-MK2 cells were mock-infected (lane 7) or infected with the indicated BHPIV3 vector (lanes 1-6) at an input multiplicity of infection of 3 TCID50 per cell. Total intracellular RNA was isolated 72 h after infection, electrophoresed on a 1% agarose gel in the presence of 0.44 M formaldehyde, transferred to nylon membrane, and hybridized with α-32P-labeled double-stranded DNA probes specific for SARS S, M, E, and N, or BPIV3 N, as indicated on the left. The images were prepared with a PhosphorImager.
Fig. 3.
Fig. 3.
Expression of SARS-CoV proteins by BHPIV3 vectors. LLC-MK2 cells were infected with the individual BHPIV3 vectors (lanes 1-7) at an input multiplicity of infection of 5 TCID50 per cell. In addition, Vero cells were mock-infected (lane 9) or infected with 5 TCID50 per cell of SARS-CoV (lane 8). Cell lysates were prepared 42 h after infection, in the case of SARS-CoV, or 72 h after infection, in the case of the BHPIV3 vectors, denatured under reducing conditions, subjected to SDS/PAGE in 4-12% gradient gels, and transferred to nitrocellulose. SARS-CoV proteins were detected by using convalescent serum from an African green monkey that had been infected with SARS-CoV. The BHPIV3 F protein was detected in a parallel blot by using HPIV3-specific rabbit hyperimmune serum (Lower). Detection of bound antibodies was done with horseradish peroxidase-conjugated goat anti-human (in the case of the anti-SARS-CoV serum) or anti-rabbit (in the case of the anti-HPIV3 serum) antibody, respectively, and visualized by chemiluminescence.
Fig. 4.
Fig. 4.
Indirect immunofluorescence of LLC-MK2 cells infected with BHPIV3 vectors. Cells on cover slips were infected at an input multiplicity of infection of 0.05 TCID50 with the BHPIV3 vectors listed to the left, incubated for 24 h, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100. SARS-CoV proteins were visualized (Left) by incubation with convalescent serum from a SARS-CoV-infected African green monkey followed by an Alexa488-conjugated goat anti-human antibody (Molecular Probes). BHPIV3 proteins were visualized (Center) by incubation with convalescence serum from HPIV3-infected hamsters, followed by Alexa594-conjugated goat anti-hamster antibody (Molecular Probes). Nuclear chromatin staining (blue) was performed with 4′,6-diamidino-2-phenylindole (Sigma).

Similar articles

See all similar articles

Cited by 102 articles

See all "Cited by" articles

MeSH terms

LinkOut - more resources

Feedback