Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats

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.

Fig. 1

(A) Construction of recombinant adenovirus Ad-S_N and (B) genomic structure of Ad-S_N.

Fig. 2

Expression of the S_N fragment in vitro and in vivo (Z: Ad-LacZ; N: Ad-S_N). (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-S_N or Ad-LacZ at the indicated MOIs for 48 h. Total cellular RNAs were isolated, and S_N mRNA was detected by RT-PCR amplification, with β-actin used as the internal control (S_N 624 bp, β-actin 417 bp). (B) Vero-E6 cells were infected with Ad-S_N or Ad-LacZ at 20 MOI for 48 h, then cells and the culture supernatants were collected. Alternatively, rats were injected subcutaneously with Ad-S_N 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 S_N 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

Ad-S_N 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-S_N. Arrows (↑) indicate time points of immunization. (B) Western blot analysis of S_N-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-S_N (separated by 10% SDS-PAGE) were used as antigen, and sera from rats immunized with Ad-S_N subcutaneously (Row 1) or intranasally (Row 2) were used as the primary antibodies. Bands indicate the existence of S_N-specific antibodies in the sera of immunized rats. (C) Analysis of neutralizing activities of sera from the rats immunized subcutaneously or intranasally with Ad-S_N.