Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain

Abstract

Background: Sublingual (s.l.) administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs) infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S) (rADV-S) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV.

Results: Here, we show that s.l. administration of rADV-S induced serum SARS-CoV neutralizing and airway IgA antibodies in mice. These antibody responses are comparable to those induced by intranasal (i.n.) administration. In addition, s.l. immunization induced antigen-specific CD8+ T cell responses in the lungs that are superior to those induced by intramuscular immunization. Importantly, unlike i.n. administration, s.l. immunization with rADV did not redirect the rADV vector to the olfactory bulb.

Conclusion: Our study indicates that s.l. immunization with rADV-S is safe and effective in induction of a broad spectrum of immune responses and presumably protection against infection with SARS-CoV.

Figure 1

Construction of rADV vector expressing SARS-CoV S glycoprotein and expression of S proteins in 293 T cells and bacteria. (A) The gene encoding codon-optimized SARS-S protein without helical regions, transmembrane domain and cytoplasmic domain was inserted into pShuttle vector to construct the rADV expressing the SARS-S protein. (B) 293 T cells were infected with rADV-S or rADV-EGFP and the S protein in the cell lysate was detected by Western blot. (C) DNA for SARS-S protein (amino acids 201–510) was inserted into pET15b vector to express recombinant S protein from E. coli. The protein was purified by His-tag affinity chromatography and detected by Western blot.

Figure 2

SARS-CoV S-specific humoral immune responses in the immunized mice. Mice were immunized with rADV-S by s.l., i.n., or i.m. route. Sera were collected two weeks after each vaccination and BAL was collected two weeks after the last immunization. S-specific IgG titers in sera (A) and S-specific IgA titers in BAL (B) were determined by ELISA. The results are expressed as the means + SD. The data are representative of three separate experiments.

Figure 3

SARS-CoV neutralizing activity of Sera. Mice were immunized three times with rADV-S 2 × 10⁷ PFU by s.l., i.n., or i.m. route. Sera were tested to measure the Abs that would neutralize the infectivity of 100 TCID₅₀ of SARS-CoV in Vero cell monolayers. Neutralization titers were determined by the CPE of SARS-CoV on Vero cell. The dotted line indicates the limitation of detection. The results are expressed as the means + SD.

Figure 4

Detection of S_366-374epitope-specific and IFN-γ secreting CD8⁺T lymphocytes in the mice vaccinated with rADV-S. Mice were immunized three times with rADV-S 1 × 10⁸ PFU by s.l., i.n., or i.m. route. Lung (A) and spleen (B) lymphocytes were harvested 10 days after the final immunization and were tested by flow cytometric analysis after staining with S_366-374 tetramer, IFN-γ, CD8 and CD44.