Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by b-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection. (Marra et al., 2003;Rota et al., 2003) and by experimental infection of macaques to fulfil Koch's postulates . 0008-1579 G 2006 SGM Printed in Great BritainCurrently, there is no effective treatment for SARS. Prevention through contact-reduction or transmission-blocking measures has been the only means available to modify the devastating impact of this illness. Prevention through vaccination would be an attractive alternative that is less reliant on individual case detection to be effective. No vaccines are currently licensed for any of the human CoVs, but effective vaccines have been produced for some animal CoVs, such as certain strains of Infectious bronchitis virus (poultry), Bovine coronavirus and Canine coronavirus (Cavanagh, 2003;Enjuanes et al., 1995;Pratelli et al., 2003;Saif, 2004;Takamura et al., 2002). Individuals convalescing from SARS develop high titres of neutralizing antibodies (Tan et al., 2004) and the appearance of antibodies coincides with the onset of resolution of SARS pneumonia Woo et al., 2004). Thus, there is some optimism that an effective vaccine against SARS-CoV may also be possible.Coronavirus spike (S) proteins have long been known to be a major determinant in coronavirus pathogenesis, given that this viral protein interacts with cellular receptors as well as con...

Rhesus and cynomolgus macaques were challenged with 107 PFU of a clinical isolate of the severe acute respiratory syndrome (SARS) coronavirus. Some of the animals developed a mild self-limited respiratory infection very different from that observed in humans with SARS. The macaque model as it currently exists will have limited utility in the study of SARS and the evaluation of therapies.A novel coronavirus was recently identified as the causative agent of severe acute respiratory syndrome (SARS) (3, 7). One factor that led to this conclusion was the demonstration by Fouchier et al. and Kuiken et al. that exposure of the SARS coronavirus (SARS CoV) to a related host (i.e., cynomolgus macaques) led to the development of a comparable disease (1, 4). In their studies, SARS CoV administered intratracheally to effect delivery to the lower respiratory tract and conjunctiva of cynomolgus macaques resulted in respiratory and constitutional symptoms, shedding of virus, and pulmonary pathology. The availability of an authentic animal model for SARS is essential for a greater understanding of pathogenesis as well as the development and evaluation of effective vaccines and pharmacologic therapies.Our studies included a 12-to 14-day life phase of evaluation with sequential clinical and virologic analyses, followed by a thorough evaluation of tissues harvested at necropsy. This differed from the previous studies, which emphasized short-term necropsies (i.e., 4 to 6 days) and end points of pathology. Individually housed macaques were acclimated in a biosafety level 3 facility prior to initiation of the experiment.Cynomolgus macaques (Philippine origin and captive bred, 1.5 to 2.0 kg) and rhesus macaques (Indian origin and captive bred, 2.9 to 4.9 kg) were administered 10 7 PFU of Tor2 SARS CoV by direct instillation into the trachea via an endotracheal tube or intravenous infusion via a saphenous vein. These experiments were approved by the Animal Care and Use Committees of the University of Pennsylvania and the Southern Research Institute. All in vivo and in vitro experiments with SARS CoV were performed under biosafety level 3 with investigators using respirators. The challenge stock was 1 passage removed from the passage 2 stock provided to us from Heinz Feldmann (Winnipeg, Canada). An aliquot of the passage stock was converted to DNA with reverse transcriptase (RT), and open reading frames spanning N, M, E, and S were PCR amplified and subcloned, and one clone from each open reading frame was sequenced (both strands to 99.9% confidence level). The nucleotide sequences of the cloned open reading frames were 99.9% identical to the published Tor2 sequence (5). The abundance of SARS CoV RNA was quantitated in tissues by TaqMan PCR using oligonucleotides or probe to N. To minimize cross-contamination between samples during the necropsy, disposable tools were used for tissue and sample processing. In the TaqMan assay, cloned SARS CoV gene fragments were used to establish standard curves. Samples not treated with RT were incorpora...

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Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.The genome sequence of severe acute respiratory syndrome (SARS) coronavirus (CoV) provided direct evidence that a new CoV is responsible for the recent SARS epidemic in Asia and Canada (11,15). This new CoV appears to have host specificities different from those of previously known CoVs. One of the potential cell tropism determinants is the spike (S) protein that recognizes the host receptor (2, 4, 21). The genome sequence of the SARS CoV predicts an S protein of 1,255 amino acids (aa) (GenBank accession no. 29836496) with 23 potential N-linked glycosylation sites (11,15), and the N terminus of the S protein contains a signal peptide that is presumably removed in the mature virion (11,15). Unlike some CoVs, such as human CoV (HCoV) 229E, in which the S protein is cleaved into S1 and S2 subunits, the S protein in SARS CoV is not cleaved. Alignment of the SARS CoV S protein with the S proteins of other CoVs, including HCoV 229E, showed low homology. However, the SARS CoV S protein could be aligned in the S2 region with higher homology than with the S1 region. S2 contains the membrane-anchoring and fusion region, which plays a key role in virus assembly and entry (8). Despite the lack of significant homology with the S1 protein of HCoV 229E, transmissible gastroenteritis virus (TGEV), or mouse hepatitis virus (MHV), which contains the receptor binding site (1,3,10,16), it is likely that the receptor binding site of the SARS CoV S protein is located in the N-terminal part corresponding to the S1 region.The host receptor for group I CoVs, such as HCoV 229E and TGEV, is aminopeptidase N (also known as CD13) (4, 21). MHV, a group II CoV (5, 6), recognizes carcinoembryonic antigen-related cell adhesion molecules as another group of CoV receptors. Viral entry is initiated by attachment of the S protein to the specific host receptor, which triggers a conformational change in the S protein. This receptor-induced conformational change exposes the fusogenic region embedded in the S2 region that inserts into the cellular membrane (12,22). In HCoV 229E, the receptor binding domain has been narrowed to a region comprising residues 407 to ...

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage. INTRODUCTIONSevere acute respiratory syndrome (SARS) caused 8098 reported cases and 774 deaths in 26 countries (WHO, 2004a) in a single autumn-to-spring period from 2002 to 2003, and had significant effects on the global economy. Serological evidence suggests zoonotic transmission of SARS coronavirus (CoV) into the human population for several years before this outbreak (Zheng et al., 2004); transmission to humans has continued, resulting in at least four independent non-laboratory associated cases in (Che et al., 2006Fleck, 2004; Guan et al., 2005; WHO, 2004b). The aetiological agent of SARS has been identified as a novel human CoV by sequencing of its genome (Marra et al., 2003;Rota et al., 2003) and by experimental infection of macaques to fulfil Koch's postulates (Fouchier et al., 2003). It is of particular concern as a zoonosis because it can replicate in a large number of animals including cats, pigs, ferrets, foxes, monkeys and rats (Chen et al., 2005; et al., 1996;Olsen, 1993). Although antibodies to CoV N proteins have no virus-neutralizing activity, there is evidence that the protein may provide protection in vivo by induction of cell-mediated immunity, although it has also been suggested to induce eosinophilic infiltrates resulting in immunopathology (Deming et al., 2006;Enjuanes et al., 1995; Stohlman et al., 1995;Wesseling et al., 1993). N protein has been shown to generate CoV-specific CD8 + T cells (Boots et al., 1991;Seo et al., 1997; Stohlman et al., 1993 Stohlman et al., , 1995; it also provides protection in animals in response to infection by animal CoV (Collisson et al., 2000;Seo et al., 1997). In addition, vaccination with SARS N protein decre...