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Mucosal Immunization with Surface-Displayed Severe Acute Respiratory Syndrome Coronavirus Spike Protein on Lactobacillus casei Induces Neutralizing Antibodies in Mice

Jong-Soo Lee,^1, Haryoung Poo,^2, Dong P. Han,³ Seung-Pyo Hong,¹ Kwang Kim,¹ Michael W. Cho,³ Eun Kim,⁵ Moon-Hee Sung,^1,4* and Chul-Joong Kim^5*

Bioleaders Corporation, Daejeon, Korea,¹ Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea;,² Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio,³ Department of Bio and Nanochemistry, College of Natural Sciences, Kookmin University, Seoul, Korea,⁴ National Research Laboratory (NRL), College of Veterinary Medicine, Chungnam National University, Daejeon, Korea⁵

Received 27 July 2005/ Accepted 16 January 2006

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.

Jong-Soo Lee and Haryoung Poo contributed equally to this work.

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