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. 2006 Sep 15;353(1):6-16.
doi: 10.1016/j.virol.2006.03.049. Epub 2006 Jun 21.

Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus S protein elicits neutralizing antibodies: Implication for developing SARS vaccines

Lanying Du  1 , Yuxian He, Yijia Wang, Haojie Zhang, Selene Ma, Charlotte K L Wong, Sharon H W Wu, Fai Ng, Jian-Dong Huang, Kwok-Yung Yuen, Shibo Jiang, Yusen Zhou, Bo-Jian Zheng
Affiliations

Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus S protein elicits neutralizing antibodies: Implication for developing SARS vaccines

Lanying Du et al. Virology. .

Abstract

Development of an effective vaccine for severe acute respiratory syndrome (SARS) remains to be a priority to prevent possible re-emergence of SARS coronavirus (SARS-CoV). We previously demonstrated that the receptor-binding domain (RBD) of SARS-CoV S protein is a major target of neutralizing antibodies. This suggests that the RBD may serve as an ideal vaccine candidate. Recombinant adeno-associated virus (rAAV) has been proven to be an effective system for gene delivery and vaccine development. In this study, a novel vaccine against SARS-CoV was developed based on the rAAV delivery system. The gene encoding RBD was cloned into a pAAV-IRES-hrGFP plasmid. The immunogenicity induced by the resulting recombinant RBD-rAAV was evaluated in BALB/c mice. The results demonstrated that (1) a single dose of RBD-rAAV vaccination could induce sufficient neutralizing antibody against SARS-CoV infection; (2) two more repeated doses of the vaccination boosted the neutralizing antibody to about 5 times of the level achieved by a single dose of the immunization and (3) the level of the antibody continued to increase for the entire duration of the experiment of 5.5 months. These results suggested that RBD-rAAV is a promising SARS candidate vaccine.

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Figures

Fig. 1
Fig. 1
Schematic diagram of S protein of SARS-CoV and pRBD-AAV. The structure of SARS-CoV S protein (A). In the S1 domain, SP and RBD range from 1 to 13 aa (13 aa) and from 318 to 510 aa (193 aa), respectively. The S2 domain contains functional domains HR1, HR2 and TM. DNA encoding 24 aa of the CD5 signal peptide and 193 aa RBD of SARS-CoV S protein was amplified and ligated into the pAAV-IRES-hrGFP plasmid to construct the recombinant pRBD-AAV vector (B).
Fig. 2
Fig. 2
RBD-rAAV production in 293T cells. After 293T cells were transfected by pRBD-AAV (A) or blank pAAV (B) together with their helper plasmids for 72 h, intense fluorescence of hrGFP was observed under fluorescence microscopy (40×).
Fig. 3
Fig. 3
Detection of RBD-rAAV transduction efficiency by fluorescence microscopy and flow cytometry analysis. The infection of RBD-rAAV and blank AAV in 293T (A) and HeLa cells (B) was detected at 72 h post-infection under fluorescence microscopy (100×). The infection rates of the AAVs in these two cell lines were further measured at 72 h post-infection by a flow cytometry analysis (C). The top right of each histogram shows the mean percentage of GFP positive cells in analyzed cell populations from two independent experiments. Black lines indicate the non-infected cells and red lines indicate the blank AAV-infected cells, while blue lines indicate the RBD-rAAV infected cells. The infection rates of RBD-rAAV and blank AAV in 293T (a) and HeLa cells (b) were further tested at different time points from 1 to 4 days (D).
Fig. 4
Fig. 4
Detection of the RBD expression in RBD-rAAV infected cells by an immunofluorescence assay and Western blot analysis. Expression of RBD in RBD-rAAV (A) and blank AAV (B) infected HeLa cells were tested by an immunofluorescence assay. The RBD expression was also detected by Western blot (C). Lanes 1 and 3, RBD-rAAV-infected HeLa and 293T cell lysates; lanes 2 and 4, blank AAV-infected HeLa and 293T cell lysates. The molecular marker (kDa) is indicated on the left. The molecular mass of the expressed RBD protein was about ∼34 kDa.
Fig. 5
Fig. 5
Evaluation of specific antibody responses in single dose of RBD-rAAV-vaccinated mice. The mice were i.m.-immunized respectively with one dose of RBD-rAAV, blank AAV and inactivated SARS-CoV with (In-SCoV + Alum) or without (In-SCoV) Alum. Serum samples were collected from the mice at pre-vaccination (0 month), 1, 2 and 4 months post-immunization. SARS-CoV-specific antibodies in the serum samples were detected by ELISA using the commercial SARS-CoV antibody detection kit (A). The neutralizing antibody titers in the sera were measured by a neutralization assay (B). The experiments were repeated four times for the RBD-rAAV group and two times for the other groups. The data were presented as the mean values ± SE.
Fig. 6
Fig. 6
Detection of specific antibody responses in three doses of RBD-rAAV-vaccinated mice. SARS-CoV-specific antibody responses in mice vaccinated with three doses of RBD-rAAV or controls were tested by ELISA (A) and neutralization assay (B). Serum samples were collected from the mice at pre-vaccination (0 month), 1, 2, 3, 4 and 5.5 months post-immunization. The experiments were repeated two times and the data were presented as the mean values ± SE.
Fig. 7
Fig. 7
Inhibitory test of RBD binding to ACE2. Sera obtained from five mice vaccinated with 3 doses of RBD-rAAV (labeled as M1–5) at 4 months post-vaccination were detected for the inhibitory effect on the binding of RBD to ACE2 by ELISA. Sera from five mice immunized with 3 doses of blank AAV were applied to the assay as the negative control. The serum samples were tested in duplicate and the data presented here were the mean values of the two tests.
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