Induction of mucosal immunity may be important for preventing SARS-CoV infections. For safe and effective delivery of viral antigens to the mucosal immune system, we have developed a novel surface antigen display system for lactic acid bacteria using the poly-␥-glutamic acid synthetase A protein (PgsA) of Bacillus subtilis as an anchoring matrix. Recombinant fusion proteins comprised of PgsA and the Spike (S) protein segments SA (residues 2 to 114) and SB (residues 264 to 596) were stably expressed in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses, immunofluorescence microscopy, and flow cytometry. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by enzyme-linked immunosorbent assays using S protein peptides. More importantly, these antibodies exhibited potent neutralizing activities against severe acute respiratory syndrome (SARS) pseudoviruses. Orally immunized mice mounted a greater neutralizing-antibody response than those immunized intranasally. Three new neutralizing epitopes were identified on the S protein using a peptide neutralization interference assay (residues 291 to 308, 520 to 529, and 564 to 581). These results indicate that mucosal immunization with recombinant L. casei expressing SARS-associated coronavirus S protein on its surface provides an effective means for eliciting protective immune response against the virus.Severe acute respiratory syndrome (SARS) recently emerged as a zoonotic infectious disease with high morbidity and mortality. The causative agent, SARS-associated coronavirus (SARSCoV), belongs to the Coronaviridae, a family of enveloped viruses containing a single-stranded RNA genome ranging from 27 to 32 kb in length (22). SARS-CoV is transmitted through the mucosal surfaces of the upper respiratory or gastrointestinal tracts, leading to pneumonia and enteritis in humans, with a limited occurrence of systemic diseases (14,18,19,27). Recent studies have identified the spike (S) protein as a major target for vaccine development because of its ability to induce neutralizing antibodies (8,10,31,33,36). Several vaccine strategies have been examined for the prevention of SARS infection, including whole inactivated virus, DNA vaccine, and recombinant viral vectors based on adenovirus, vaccinia virus, and parainfluenza virus (3,4,11,20,35).Studies of the development of animal coronavirus vaccines have demonstrated that systemic humoral or cell-mediated immune responses induced by parenteral administration may not be enough to prevent infection in mucosal areas (17). The prominent role of the mucosa in SARS transmission and infection suggests that direct mucosal immunization could be an effective strategy for prophylaxis by induction of systemic and mucosal immune responses. For mucosal immunization, lactic acid bacteria (LAB) are more attractive as delivery vehicles than other live-vaccine vectors (e.g., Shigella, Salmonell...