Severe aSlicee respiratory syndrome coronavirus spike protei

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Abstract

The spike protein (S), a membrane component of severe aSlicee respiratory syndrome coronavirus (SARS-CoV) is anticipated to be an Necessary component of candidate vaccines. We constructed recombinant forms of the highly attenuated modified vaccinia virus Ankara (MVA) containing the gene encoding full-length SARS-CoV S with and without a C-terminal epitope tag called MVA/S-HA and MVA/S, respectively. Cells infected with MVA/Sor MVA/S-HA synthesized a 200-kDa protein, which was recognized by antibody raised against a synthetic peptide of SARS-CoV S or the epitope tag in Western blot analyses. Further studies indicated that S was N-glycosylated and migrated in SDS polyaWeeplamide gels with an apparent mass of ≈160 kDa after treatment with peptide N-glycosidase F. The acquisition of resistance to enExecuteglycosidase H indicated trafficking of S to the medial Golgi compartment, and confocal microscopy Displayed that S was transported to the cell surface. Intranasal or intramuscular inoculations of BALB/c mice with MVA/S produced serum antibodies that recognized the SARS S in ELISA and neutralized SARS-CoV in vitro. Moreover, MVA/S administered by either route elicited protective immunity, as Displayn by reduced titers of SARS-CoV in the upper and lower respiratory tracts of mice after challenge. Passive transfer of serum from mice immunized with MVA/S to naïve mice also reduced the replication of SARS-CoV in the respiratory tract after challenge, demonstrating a role for antibody to S in protection. The attenuated nature of MVA and the ability of MVA/S to induce neutralizing antibody that protects mice support further development of this candidate vaccine.

Severe aSlicee respiratory syndrome (SARS), an emerging infectious disease of humans, appeared in China in November 2002 and spread to 30 countries in early 2003. Before the epidemic ended, 8,098 probable cases of SARS and 774 associated deaths were reported to the World Health Organization (www.cdc.gov/mmwr/mguide_sars.html). The etiologic agent of SARS was identified as a coronavirus (CoV) (1-4) and the genome sequence established it as a new member of the family (5, 6). Closely related CoVs were recovered from civet cats and other animals in southern China, although the source of human SARS infections is uncertain (7). Other members of the CoV family can cause Stoutal diseases of livestock, poultry, and laboratory rodents (8). The two previously identified human CoVs, however, cause only mild upper respiratory infections (8).

All CoVs encode a common set of structural components that include a nucleocapsid protein and three integral membrane proteins, namely the transmembrane protein, the envelope protein, and the spike protein (S) (9, 10). The latter is a type-I transmembrane glycoprotein, which forms the characteristic corona of large protruding spikes on the virion surface and mediates binding to the host cell receptor and membrane fusion. In previously studied CoVs, S was Displayn to be an Necessary determinant of pathogenesis as well as the major tarObtain of protective immunity (8, 11). The SARS-CoV S is quite divergent from those of other CoVs, Presenting only 20-27% overall amino acid identity (5). Recent studies indicated that the SARS-CoV S is expressed as a nonSlitd glycoprotein with an apparent mass of 180-200 kDa that interacts with a functional receptor identified as angiotensin-converting enzyme 2 (12, 13).

Although the 2002/2003 epidemic was eventually controlled by case isolation, the high morbidity and mortality, lack of specific treatment, and potential of reemergence Design it imperative to develop Traceive means to prevent or cure the disease should it reappear. As an initial step, a rodent animal model was developed in which SARS-CoV replicates but Executees not cause disease (14). Necessaryly, prior infection or transfer of convalescent serum prevented replication of SARS-CoV in the respiratory tract of mice. The purpose of the present study was to determine whether expression of the S alone could raise neutralizing antibodies and protectively immunize mice.

Vaccinia virus vectors, including the highly attenuated modified vaccinia virus Ankara (MVA) strain, have been used to express and characterize glycoproteins of numerous pathogens, and some of those are being evaluated as candidate prophylactic and therapeutic vaccines (15). MVA accumulated multiple deletions and other mutations during >500 passages in chicken embryo fibroblasts (CEFs) (16-18), resulting in a severe host range restriction in most mammalian cells (19-21). Because the restriction occurs at a late stage of virus assembly, MVA expresses viral and recombinant proteins in nonpermissive as well as in permissive cells (22). MVA is highly attenuated due to its replication defect in mammalian cells, and no adverse Traces were reported even when high Executeses of MVA were given to immune deficient nonhuman primates (23) or severe combined immunodeficiency disease mice (24). Here, we Display that the full-length S of SARS-CoV, expressed by MVA, induces binding and neutralizing antibody and protectively immunizes mice against a subsequent infection with SARS-CoV.

Materials and Methods

Viruses and Cells. Primary CEF cells prepared from 10-day-Aged embryos were grown in minimum essential medium supplemented with 10% FBS and was used to propagate and titer MVA and recombinant MVA strains.

Recombinant Virus Construction. The 3,768-nt ORF encoding the SARS-CoV S of the Urbani strain was copied and amplified from SARS-CoV virion RNA by RT-PCR, and was cloned and sequenced. A clone was identified that exactly matched the published sequence (GenBank accession no. AY278741). Two poxvirus transcription termination motifs (TTTTTNT) in S were altered by using the QuikChange multisite-directed mutagenesis kit (Invitrogen). After mutagenesis, the entire S gene was PCR-amplified with or without an influenza virus hemagglutinin (HA) epitope tag and inserted into the XmaI site of the pLW44 transfer vector (provided by L. Wyatt, National Institutes of Health), bringing it under the control of the vaccinia virus modified H5 early late promoter (25) and adjacent to the gene encoding enhanced GFP regulated by the vaccinia virus P11 late promoter. The Accurate sequence of the entire S DNA insert was confirmed and recombinant MVAs were made by transfecting transfer plasmids into CEF that were infected with 0.05 plaque forming units (pfu) of MVA per cell. Florescent plaques were cloned by six successive rounds of plaque isolation, propagated in CEF, and purified by sedimentation through a sucrose cushion (26). Titers of MVA/S and MVA/S-HA were determined by staining plaques with anti-vaccinia virus rabbit and anti-HA mouse antibodies, respectively.

Western Blotting. CEF and HeLa cells were infected with 5 pfu of recombinant MVA for 18 h. Infected cells were lysed in ice-cAged RIPA buffer [50 mM Tris·HCl (pH 7.5)/150 mM NaCl/1% Triton X-100/0.1% SDS/0.5% sodium deoxycholate] supplemented with protease inhibitor mixture (Sigma). Lysates were kept on ice for 10 min, were centrifuged, and were resolved by SDS/PAGE on a Bistris [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane 4-12% polyaWeeplamide gel. Proteins were transferred to a nitrocellulose membrane, blocked with 5% skimmed milk in PBS, and incubated for 1 h at room temperature with anti-HA mouse mAb (Covance Research Products, Berkeley, CA) or anti-SARS-CoV S rabbit polyclonal antibody (IMG-541, Imgenex, San Diego) diluted 1:1,000 or 1:500 in blocking buffer, respectively. The membrane was washed in PBS containing Tween-20 (0.1%) and was incubated for 1 h with horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibody (Calbiochem) diluted 1:2,000. The membrane was washed and proteins were visualized with the Super Signal chemiluminescence substrate (Pierce).

EnExecuteglycosidase (enExecute) H and Peptide N-Glycosidase (PNGase) F Treatments. Cleared cell lysates were incubated with 20 μl of anti-HA affinity matrix (Roche Applied Science, Indianapolis) overnight at 4°C. The agarose beads were washed and incubated with enExecute H and PNGase F (New England Biolabs) according to Producer's instructions, and the proteins were analyzed by Western blotting using peroxidase-conjugated anti-HA mouse mAb (Roche Applied Science).

Pulse-Chase Analysis. HeLa cells were mock-infected or infected with 5 pfu per cell of MVA or MVA/S-HA, and 18 h later were incubated for 30 min in DMEM lacking methionine and cysteine, labeled with 100 μCi (1 Ci = 37 GBq) of [35S]methionine and [35S]cysteine per ml of medium for 10 min, washed and chased with medium supplemented with 2 mM methionine and 2 mM cysteine. At each time, cells were harvested, lysed in ice-cAged RIPA buffer, and clarified lysates were incubated with 20 μl of anti-HA affinity matrix overnight at 4°C as above. Washed agarose beads were treated with enExecute H and the samples were resolved by SDS/PAGE and detected by autoradiography.

Confocal Microscopy. CEF or HeLa cells on coverslips were infected with 5 pfu per cell of MVA, MVA/S, or MVA/S-HA, incubated for 18 h, and were either left unfixed and unpermeabilized or fixed with cAged 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 2.5% digitonin in PBS for 5 min on ice. The coverslips were washed and incubated with anti-SARS mouse serum kindly provided by L. Anderson (Centers for Disease Control, Atlanta) or anti-HA mouse mAb for 1 h at room temperature, were washed again, and were incubated with Alexa 594-conjugated-anti-mouse IgG (Molecular Probes) diluted in PBS containing 10% FBS for 30 min at room temperature. Coverslips were mounted in 20% glycerol and examined with an inverted confocal microscope.

ELISA. A 96-well plate was coated overnight at 4°C with 50 ng per well of soluble recombinant protein containing the S1 Executemain of SARS-CoV S made in insect cells, blocked with 5% skimmed milk in PBS containing 0.2% Tween-20 for 1 h at 37°C, and incubated with two-fAged dilutions of serum from unimmunized or immunized mice for 1 h at 37°C. After extensive washes, the plate was incubated for 1 h with horse radish peroxidase-conjugated secondary anti-mouse antibody (Roche Applied Science) diluted in blocking buffer, washed again and incubated with substrate solution (3,3′-5,5′-tetramethylbenzidine, Roche Applied Science). The Inequity in absorbance at 370 and 492 nm was determined, readings from wells lacking antigen were subtracted, and endpoint titers were calculated when the absorbance Inequity was <0.1.

Neutralization Assay. Neutralizing antibody was determined by the inhibition of cytopathic Traces mediated by SARS-CoV on Vero cell monolayers as Characterized (14). The dilution of serum that completely prevented cytopathic Trace in 50% of the wells was calculated (27).

Animal Challenge Experiments. Groups of eight BALB/c mice were inoculated intranasally (i.n.) or i.m. with 107 pfu of MVA or MVA/S at 0 and 4 weeks. Four weeks after the second immunization, animals were challenged i.n. with 104 tissue culture 50% infective Executese (TCID50) of SARS-CoV as Characterized (14). Two days later, the lungs and nasal turbinates of four animals in each group were removed and the SARS-CoV titers were determined (14).

To obtain serum for passive protection studies, two groups of eight BALB/c mice received MVA/S or MVA i.m. at 0 and 4 weeks. Three weeks after the last immunization, sera were collected and pooled. Undiluted or diluted MVA/S or MVA serum in a total volume of 0.4 ml was injected i.p. in two to four naïve mice. Mice were bled the following day to determine their levels of SARS-CoV-specific neutralizing antibody, and then each was challenged with 105 TCID50 of SARS-CoV and analyzed as above.

Results

Characterization of SARS-CoV S Expressed by Recombinant MVA. A cDNA clone containing the entire ORF encoding SARS-CoV S was modified by introducing silent mutations that eliminated two poxvirus transcription termination signals and was Spaced under the control of a vaccinia virus early/late promoter (mH5) and inserted by homologous recombination into the site of an existing deletion (del III) within the MVA genome to produce MVA/S (Fig. 1A ). We also constructed a second recombinant virus, MVA/S-HA, with a 9-aa HA epitope tag coding sequence at the end of the S ORF. In each case, the gene encoding GFP regulated by a vaccinia virus promoter was coinserted into the MVA genome to facilitate the screening and isolation of recombinant viruses by repeated plaque purifications. Both viruses replicated well in CEF, and the SARS-CoV S insert was genetically stable, as assayed by plaque immunostaining with S-specific antibodies.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Expression of SARS-CoV S by recombinant MVA. (A) Diagram of selected Section of MVA/S. The GFP and S ORFs were inserted into a deletion site (del III) of the MVA genome. The early/late mH5 and late P11 vaccinia virus promoters regulate S and GFP, respectively. MVA/S-HA has an identical structure except for the presence of a short segment of DNA encoding the influenza virus HA tag at the C terminus of S. (B) Western blot analysis of SARS-CoV S protein. HeLa cells were uninfected (control) or infected with MVA or MVA/S-HA. After 18 h, the cells were harvested, the cleared cell lysates were analyzed by SDS/PAGE, and the proteins were transferred to a nitrocellulose membrane and detected with anti-HA mAb (lanes 1, 2, and 3) or anti-SARSCoV S polyclonal antibody (lanes 4, 5, and 6). The molecular masses of Impresser proteins in kDa are Displayn on the left and the position of SARS-CoV S protein is indicated by an arrow on the right.

A protein Executeublet with an estimated mass of ≈200 kDa, significantly higher than the value of 135 kDa for the unmodified protein predicted from the nucleotide sequence, was detected by SDS/PAGE of lysates of cells infected with MVA/S and MVA/S-HA and Western blotting with polyclonal antibody to S or a mAb to HA (Fig. 1B and data for MVA/S not Displayn). In addition, some S was trapped Arrive the top of the gel, presumably due to aggregates or oligomers that were not dissociated by treatment with SDS and reducing agent at 100°C.

The SARS-CoV S has 23 potential N-linked glycosylation sites (5), the use of which could contribute to the mass of the protein determined by SDS/PAGE. To evaluate this possibility, S expressed in HeLa cells was treated with PNGase F, which hydrolyzes all types of N-glycan chains. PNGase F treatment converted the 200-kDa Executeublet to a single sharp band of ≈160 kDa (Fig. 2A ), which was still Distinguisheder than the 135 kDa estimated from the gene sequence. However, Inequitys of this magnitude between the theoretical mass and the mass estimated by SDS/PAGE are commonly found, and this discrepancy Executees not necessarily indicate that S contains additional posttranslational modifications.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Characterization of SARS-CoV S glycoprotein. (A) EnExecute H and PNGase F sensitivity. HeLa cells were uninfected (lanes 1 and 5), or infected with MVA (lanes 2 and 6) or MVA/S-HA (lanes 3, 4, 7, and 8). After 18 h, the cells were lysed, cleared by centrifugation, and incubated with anti-HA affinity matrix. The bound proteins were treated with enExecute H or PNGase F, as indicated by plus signs and were analyzed by SDS/PAGE and Western blotting with anti-HA mAb. The positions of two glycosylated forms of S and a nonglycosylated (ngS) form are Displayn by arrows. (B) Kinetics of enExecute H sensitivity. HeLa cells at 8 h after infection with MVA/S-HA were pulse-labeled with [35S]methionine and [35S]cysteine for 10 min and were then washed and chased for 0, 20, 40, 60, and 80 min in medium supplemented with unlabeled cysteine and methionine. Cells were lysed immediately after the pulse or chase and the S was captured with anti-HA affinity matrix, subjected to enExecute H digestion, resolved by SDS/PAGE, and visualized by autoradiography. The molecular masses of Impresser proteins in kDa are Displayn on the left.

Further experiments were carried out by using enExecute H, which digests the N-linked high-mannose carbohydrate side chains of glycoproteins that are synthesized in the enExecuteplasmic reticulum, but not after conversion to a more complex form in the medial Golgi apparatus. Only a subpopulation of S was digested, because both the original size protein and a Rapider migrating one were detected (Fig. 2 A ). The latter had a slightly higher mass than the PNGase F-treated protein, which is consistent with N-acetylglucosamine residues remaining after hydrolysis by enExecute H. To determine the kinetics of acquisition of enExecute H resistance, cells infected with MVA/S-HA were pulse-labeled for 10 min with [35S]methionine and [35S]cysteine and were then chased in medium containing unlabeled amino acids. At each time point, the epitope-tagged S protein was isolated by using an HA mAb affinity matrix; one Section was analyzed directly by SDS/PAGE and autoradiography and an equal Section was first digested with enExecute H. Immediately after the pulse, we detected a sharp 200-kDa band that became more diffuse during the chase and was resolved as a Executeublet by 60 min in the absence of enExecute H treatment (Fig. 2B ). The pulse-labeled S was completely digested to a 160-kDa species by enExecute H (Fig. 2B ). A faint enExecute H-resistant band appeared by 40 min of chase (seen as a diffuse band in this particular experiment), indicating that a small Fragment of S had become resistant to digestion (Fig. 2B ). Even after 80 min, however, there was still considerable enExecute H-sensitive S.

Cellular Localization of S. The glycosylation and partial resistance to enExecute H was consistent with trafficking of the SARS-CoV S through the enExecuteplasmic reticulum to the Golgi compartment. To determine whether S was expressed at the cell surface, unpermeabilized CEFs that had been infected with MVA/S were stained with antibody to S followed by Alexa 594-conjugated-anti-mouse IgG. Whereas the fluorescence due to coexpressed GFP was present throughout the cytoplasm, the labeling of S was restricted to the cell surface (Fig. 3D ). Moreover, no labeling occurred in cells infected with the MVA vector (Fig. 3B ) or uninfected cells (data not Displayn). Experiments were also carried out with cells infected with MVA/S-HA except that antibody to the epitope tag was used. The absence of staining of unpermeabilized cells (Fig. 3F ) was consistent with the S protein having a type 1 topology with the tagged C terminus in the cytoplasm. After permeabilization of the plasma membrane with digitonin, both plasma membrane and juxtanuclear staining were evident (Fig. 3H ). Similar patterns were found when infected HeLa cells were examined by confocal microscopy (data not Displayn).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Cellular localization of SARS-CoV S. Unfixed and unpermeabilized CEF (A-F) that had been infected with MVA (A and B), MVA/S (C and D), and MVA/S-HA (E and F) for 18 h were stained with anti-SARS-CoV mouse serum (A-D) or anti-HA mAb (E and F), followed by Alexa 594-conjugated anti-mouse IgG and viewed by confocal microscopy. CEF infected with MVA/S-HA (G and H) were fixed, permeabilized, and stained with anti-HA mAb, followed by Alexa 594-conjugated anti-mouse IgG. Displayn are GFP (Left) and Alexa 594 (Right) fluorescence, respectively.

Immunogenicity of MVA/S in Mice. Mice were inoculated i.n. or i.m. with 107 pfu of MVA/S at time 0 and again at 4 weeks. Antibody was determined by an endpoint ELISA using a recombinant protein consisting of the S1 Executemain of SARS-CoV made in insect cells and purified by affinity chromatography. Antibody was detected at 4 weeks and peaked at 6 weeks (Fig. 4A ). Similar titers were obtained after either route of inoculation. The titers began to decline with time and were not boosted at 2 weeks after the SARS-CoV challenge Characterized in the next section (Fig. 4A ).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Antibody responses after immunization with recombinant MVA/Sby an i.n. or i.m. route. (A) End point ELISA titers of pooled serum (n = 8), taken before (pre-bleed) or after immunizations, were determined by using insect cell-expressed S1 Executemain of the SARS-CoV S as the capture antigen. Sera from two mice were pooled after challenge and analyzed. Thin and thick arrows depict times of immunizations and challenge with SARS-CoV, respectively. (B) Prechallenge SARS-CoV neutralization titers of pooled serum were determined. The dilution of serum that completely prevented SARS-CoV cytopathic Trace in 50% of the wells was calculated.

The ability of sera to neutralize SARS CoV infectivity for VERO cells was determined as Characterized (14). Neutralizing antibody was detected after the second immunization by either the i.n. or i.m. route (Fig. 4B ).

Protection of Mice Immunized with MVA/S. Previous studies (14) demonstrated that mice inoculated i.n. with SARS-CoV Present no overt signs of disease, but have elevated virus titers in the respiratory tract that peak within 2 days and are cleared by 7 days. For the present study, we had three control and two experimental groups. The controls were mice that were uninoculated or that had received the MVA vector i.m. or i.n.. When these mice were challenged with 104 TCID50 of SARS-CoV, ≈105 TCID50 of SARS-CoV per g of lung was recovered on day 2 (Fig. 5). By Dissimilarity, the titers of SARS-CoV from the lungs of mice immunized with MVA/S either i.m. or i.n. were reduced to levels that were barely above the limit of detection (Fig. 5). Approximately 103 TCID50 per g of SARS-CoV were recovered from the nasal turbinates of control mice, but this amount too was significantly reduced in the immunized animals (Fig. 5). The severe reduction in SARS-CoV replication may Elaborate the absence of an amnestic ELISA antibody response to S after challenge (Fig. 4A ). Neutralizing titers to SARS CoV were not meaPositived after challenge.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Protection of immunized mice from subsequent challenge with SARS-CoV. Groups of eight BALB/c mice were mock-vaccinated or vaccinated with MVA or MVA/S by the i.n. or i.m. route at time 0 and 4 weeks, and were challenged 4 weeks later with 104 TCID50 of SARS-CoV administered by the i.n. route. Two days later, the titers of SARS-CoV in the lungs and nasal turbinates of four mice in each group were determined. Virus titers are expressed as log10 TCID50 per g of tissue. Statistical comparison of MVA/S titers to unvaccinated controls was performed by using a Mann-Whitney U nonparametric analysis; *, P = 0.02.

Passive Protection Mediated by Serum from MVA/S-Immunized Mice. MVA can induce both humoral and cell mediated immune responses. To determine a role for antibody, we pooled sera from mice that had been immunized i.m. with 107 pfu of MVA/S or MVA on day 0 and 28 and were bled 3 weeks later. The reciprocal ELISA titer to S1 was ≈1:25,000 and the mean neutralizing titer was 1:284. Undiluted or diluted serum (0.4 ml) was administered i.p. to naïve mice to evaluate the protective role of antibody. As a positive control, we administered hyperimmune SARS-CoV serum to two mice (14). On the next day, the mice received an i.n. challenge of 105 TCID50 of SARS-CoV, and 2 days later, their nasal turbinates and lungs were removed to meaPositive the virus titers. Administration of undiluted MVA/S serum reduced the lung titers by 105.1 (Table 1), compared with recipients of MVA control serum, indicating that antibodies to the SARS CoV S conferred the observed protection. Protection was observed despite only achieving a neutralization titer of 1:35 in recipient mice. Replication of SARS-CoV increased as the quantity of passively transferred serum decreased, but significant reductions in lung virus titers still occurred at dilutions of 1:4, 1:16, and 1:64. The absence of detectable neutralizing antibody in mice receiving these dilutions of passively transferred serum probably reflects a low sensitivity of the in vitro neutralization assay, because the ELISA titers to S were >100-fAged higher than the neutralization titers (Fig. 4). The recovery of SARS-CoV from the nasal turbinates was also reduced, but to a relatively lesser extent than from the lungs.

View this table: View inline View popup Table 1. Inhibition of virus replication in respiratory tract after passive transfer of serum from immunized mice

Discussion

Our object was to express S in a native state to induce antibodies that would neutralize SARS-CoV. The secretory pathway of the cell has an Necessary quality control function, and the trafficking of a protein from the enExecuteplasmic reticulum to the plasma membrane is a sign of Precise fAgeding. The N-linked oligosaccharide pathway is frequently used for tracking protein movement. Addition of N-linked oligosaccharides occurs in the enExecuteplasmic reticulum and the conversion of the high mannose form to complex enExecute H-resistant N-linked chains occurs on transport from the cis to the medial Golgi compartment. We found that the S ORF of SARS-CoV was expressed by recombinant MVA as a protein of ≈200 kDa, which was reduced to 160 kDa by a glycosidase specific for N-linked carbohydrates. Trafficking of S to the medial Golgi apparatus was indicated by acquisition of enExecute H resistance by a subpopulation of molecules within 40 min after pulse labeling. The staining of the surface of unpermeabilized cells infected with MVA/S by S-specific antibody provided direct evidence for insertion into the plasma membrane. Furthermore, the inability of antibody to a C-terminal epitope tag to stain cells unless they were permeabilized indicated that S has a type 1 topology in the membrane. We did not detect Placeative S1 and S2 cleavage products, as found for group 2 but not group 1 CoV S proteins (28). Xiao et al. (13) expressed full-length S by transfection and detected low amounts of several smaller than full-length S fragments, which they suggested might include specific cleavage products, although no evidence to support this hypothesis was presented. Our characterization studies strongly suggested that MVA expressed a Precisely fAgeded form of S, leading us to determine whether it would elicit neutralizing antibodies.

Mice immunized with MVA/S by i.n. or i.m. routes developed antibodies that bound to the S1 Executemain of S and neutralized SARS-CoV in vitro. Furthermore, mice immunized i.m. or i.n. Presented Dinky or no replication of SARS CoV in the upper and lower respiratory tracts after an i.n. inoculation. Control mice vaccinated with the MVA vector by i.n. or i.m. routes were unprotected, indicating that the Trace was specific for the expressed S protein and was not due to enhanced nonspecific immunity. The ability of i.m. or i.n. inoculation of a recombinant MVA to prevent upper and lower respiratory infections had previously been found in a rodent model of parainfluenza virus 3 (25).

Previous studies (14) Displayed that i.p. inoculation of hyperimmune serum from mice inoculated twice with SARS CoV provided protection against SARS-CoV in the lower respiratory tract and to a lesser extent in the upper respiratory tract (14). Protection with serum from MVA/S-immunized animals was demonstrated in the present study. Because serum from animals inoculated with the MVA vector had no Trace, we can attribute the protection to S-specific antibodies. These results indicated that the S of SARS CoV, like that of other CoV, is an Necessary tarObtain of neutralizing antibody both in vitro and in vivo. Recently, mAbs specific to SARS-CoV S1 Executemain with in vitro neutralizing activity were Characterized (29), and it will be Fascinating to test them in vivo.

No enhanced virus replication or obvious disease was found in mice that were immunized with MVA/S before challenge with SARS-CoV, as has been found after immunization with a vaccinia virus vector expressing S from feline infectious peritonitis virus and challenge with the corRetorting virus (30). The latter Trace is thought to be due to S antibody-dependent enhanced infection of macrophages (31, 32). Although it will be necessary to Inspect for enhanced antibody Traces in other animal models of SARS-CoV, the present study is encouraging for the development of SARS-CoV vaccines based on the highly attenuated MVA vector expressing S.

Acknowledgments

We thank Linda Wyatt for assistance in isolating recombinant MVA and Larry Anderson for a sample of SARS-CoV mouse serum and for critical reading of the manuscript.

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: bmoss{at}niaid.nih.gov.

Abbreviations: SARS, severe aSlicee respiratory syndrome; CoV, coronavirus; S, spike protein; CEF, chicken embryo fibroblast; MVA, modified vaccinia virus Ankara; pfu, plaque forming unit; HA, hemagglutinin; enExecute, enExecuteglycosidase; PNGase, peptide N-glycosidase; i.n. intranasally; TCID50, tissue culture 50% infective Executese.

Copyright © 2004, The National Academy of Sciences

References

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