Mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on Lactobacillus casei induces neutralizing antibodies in mice

Abstract

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-gamma-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.

FIG. 1.

Construction and expression of chimeric S proteins. (A) A schematic diagram of pHAT:pgsA-SA and pHAT:pgsA-SB. (B) Western blot analyses of PgsA-SA (top) and PgsA-SB (bottom) expression in L. casei using anti-pgsA (left) and anti-SARS S (right) polyclonal antibodies. Lanes 1 and 2 show whole-cell lysates of wild-type (parental vector) and recombinant L. casei, respectively. Lanes 3 and 4 show cytoplasmic and membrane fractions of recombinant L. casei, respectively. Protein bands of ∼55 and 79 kDa, corresponding to the expected sizes of PgsA-SA and PgsA-SB, respectively, were detected in lanes 2 and 4. (C) Fluorescence-activated cell sorter histograms of wild-type (filled) and recombinant (open) L. casei cells. The cells were probed with either polyclonal rabbit anti-PgsA (top) or anti-SARS S (middle and bottom) polyclonal antibodies, followed by biotin-conjugated anti-rabbit IgG antibody and fluorescein isothiocyanate-conjugated streptavidin. (D) Representative immunofluorescence images of wild-type (control) and recombinant L. casei cells expressing PgsA-SA and PgsA-SB. Bright-field images are shown on the left.

FIG. 2.

Systemic anti-SARS S protein response following mucosal immunization. Groups of 12 mice were immunized orally (open) or intranasally (closed) with either wild-type (circles) or a mixture of recombinant (inverted triangles) L. casei. (Left) Kinetics of anti-SARS S serum IgG response. ELISA was performed in triplicate using pooled peptides shown in Table 1, and titers are defined as the reciprocal of the maximum dilution of sera that yielded an absorbance equal to that of preimmune samples. Immunization periods are indicated by bars below the x axis. (Right) Isotype profiles of anti-SARS S serum antibodies. Serum samples collected on day 84 postimmunization were used. End point titers were calculated as the reciprocals of serum dilutions yielding the same optical density as a 1/50 dilution of a pooled preimmune serum. The data are presented as means ± standard deviations. Statistical comparisons between groups were made by the Mann-Whitney U test.

FIG. 3.

Anti-SARS mucosal IgA antibody responses. Intestinal and bronchoalveolar lavage fluids, harvested from mice sacrificed 70 or 84 days postimmunization, were analyzed by ELISA in triplicate. Optical densities (OD) of samples from animals immunized orally (A) or intranasally (B) are shown. Fluids from control animals are shown as black bars. The error bars represent standard deviations.

FIG. 4.

SARS-CoV pseudovirus-neutralizing activity. Purified IgG from sera (A) and IgA from either intestinal or bronchoalveolar lavage fluids (B) were evaluated for SARS pseudovirus-neutralizing activities. Mice immunized either orally or intranasally were sampled at the indicated number of weeks after the first immunization. Approximately 100 infectious units of pseudovirus were used. Con., control; P.C., positive control. The assays were performed in duplicate, and the graphs are representative of two independent experiments. The error bars represent standard deviations.

FIG. 5.

Identification of neutralization epitopes using a peptide neutralization interference assay. (A) Four different mixtures of overlapping peptides derived from S glycoprotein (Table 2) were evaluated for the ability to interfere with the neutralizing activity of IgG purified from sera collected 6 to 8 weeks after the first oral immunization. (B) Neutralization interference by individual peptides contained in mixtures 1 and 4 using serum IgG. The numbers indicate the amino acid residues of the S glycoprotein. The assays were performed in duplicate, and the graphs are representative of two independent experiments. The error bars represent standard deviations.

FIG. 6.

Locations of SARS neutralization epitopes identified to date. A proposed three-dimensional model of the S1 domain (aa 17 to 680; Protein Data Bank identifier, 1Q4Z) was used to plot neutralization epitopes identified by this and previously reported studies. The orientation of the molecule was arbitrarily chosen for optimal display of the epitopes. The aspartic acid residue at 454 (indicated in red), which has been shown to be critical for binding to ACE2, is shown as a reference point. Panels A and B show the opposite sides of the protein. The three epitopes identified in this study are indicated in yellow (520 to 529), purple (564 to 581), and blue (291 to 308).