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. 2005 Sep;112(1-2):24-31.
doi: 10.1016/j.virusres.2005.02.009.

Adenoviral Expression of a Truncated S1 Subunit of SARS-CoV Spike Protein Results in Specific Humoral Immune Responses Against SARS-CoV in Rats

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Free PMC article

Adenoviral Expression of a Truncated S1 Subunit of SARS-CoV Spike Protein Results in Specific Humoral Immune Responses Against SARS-CoV in Rats

Ran-Yi Liu et al. Virus Res. .
Free PMC article

Abstract

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV), but the prophylactic treatment of SARS-CoV is still under investigation. We constructed a recombinant adenovirus containing a truncated N-terminal fragment of the SARS-CoV Spike (S) gene (from--45 to 1469, designated Ad-S(N)), which encoded a truncated S protein (490 amino-acid residues, a part of 672 amino-acid S1 subunit), and investigated whether this construct could induce effective immunity against SARS-CoV in Wistar rats. Rats were immunized either subcutaneously or intranasally with Ad-S(N) once a week for three consecutive weeks. Our results showed that all of the immunized animals generated humoral immunity against the SARS-CoV spike protein, and the sera of immunized rats showed strong capable of protecting from SARS-CoV infection in vitro. Histopathological examination did not find evident side effects in the immunized animals. These results indicate that an adenoviral-based vaccine carrying an N-terminal fragment of the Spike gene is able to elicit strong SARS-CoV-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV infection.

Figures

Fig. 1
Fig. 1
(A) Construction of recombinant adenovirus Ad-SN and (B) genomic structure of Ad-SN.
Fig. 2
Fig. 2
Expression of the SN fragment in vitro and in vivo (Z: Ad-LacZ; N: Ad-SN). (A) Vero-E6 cells (2 × 105 cells/well) were seeded into a 6-well plate and cultured for 16 h until cells reached 80% confluence. Cells were then infected with Ad-SN or Ad-LacZ at the indicated MOIs for 48 h. Total cellular RNAs were isolated, and SN mRNA was detected by RT-PCR amplification, with β-actin used as the internal control (SN 624 bp, β-actin 417 bp). (B) Vero-E6 cells were infected with Ad-SN or Ad-LacZ at 20 MOI for 48 h, then cells and the culture supernatants were collected. Alternatively, rats were injected subcutaneously with Ad-SN or Ad-LacZ (1 × 107 pfu/rat), after 48 h, tissues near the injection site were sampled and homogenized. Western blotting was used to detect SN protein in the infected cells, culture supernatants and rat tissues using the Phototope-HRP Western blot detection system (New England BioLabs). Serum from convalescent SARS patients (Row 1) or rabbit anti-spike IgG, SARS spike N-term D204 antibody (Row 2) were used as the primary antibodies, with an anti-actin mAb (C-2) (Santa Cruz, sc-8432) (Row 3) as the control.
Fig. 3
Fig. 3
Ad-SN induced rats to produce high titers of antibodies (A, B) and neutralization activity (C). (A) ELISA assay for SARS-CoV-specific serum IgG in the rats immunized subcutaneously (s.c.) or intranasally (i.n.) with Ad-SN. Arrows (↑) indicate time points of immunization. (B) Western blot analysis of SN-specific antibodies in sera of immunized rats. Western blotting was performed with the Phototope-HRP Western Blot Detection System (New England BioLabs). The culture supernatants of Vero-E6 cells infected with Ad-SN (separated by 10% SDS-PAGE) were used as antigen, and sera from rats immunized with Ad-SN subcutaneously (Row 1) or intranasally (Row 2) were used as the primary antibodies. Bands indicate the existence of SN-specific antibodies in the sera of immunized rats. (C) Analysis of neutralizing activities of sera from the rats immunized subcutaneously or intranasally with Ad-SN.

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