2.2. Golden Syrian hamsters

The golden Syrian hamster (strain LVG, Charles River Laboratories; http://www.criver.com/research_models_and_services/research_models/LVG_Hamsters.html) is an excellent model for SARS-CoV infection because viral replication is accompanied by pathological changes in the lungs including pneumonitis and consolidation (Roberts et al., 2005b). Following intranasal inoculation with 10³  TCID₅₀ SARS-CoV, hamsters support viral replication in the nasal turbinates and lungs. Peak viral replication in the lungs occurs at day 3 p.i. and is cleared by day 7 p.i. Virus titers in nasal turbinates are about 10–100-fold lower than in lungs, but are detectable through day 14 p.i. Viremia is detected 2–3 days p.i.; and virus can be recovered from the spleen and liver. Histopathological changes in the lungs accompany SARS-CoV infection of hamsters. By day 3 p.i., focal areas of interstitial inflammation and consolidation are visible. At day 5 p.i., inflammation is more pronounced and consolidation is more widespread. Consolidation continues through day 7 p.i. and involves 30–40% of the surface area of the lung. By day 21 p.i., pathological changes in the lung have resolved (Roberts et al., 2005b).

Although histopathological signs of disease are pronounced, clinical symptoms of disease have been difficult to identify in hamsters. However, use of exercise wheels to evaluate overnight activity demonstrates that hamsters infected with SARS-CoV are less active from day 2 through day 7 p.i. than they were prior to infection or compared to uninfected or mock-infected animals (Table 1 ). This is the first objective evidence of clinical illness in hamsters.

2.3. Evaluation of various strains of SARS-CoV in hamsters

Several strains of SARS-CoV have been evaluated in the hamster model, including Urbani (GenBank no. AY27874), HKU-39849 (HK-39; GenBank no. AY278491), Frankfurt 1 (Frk-1; GenBank no. AY291315) and a recombinant SARS-CoV infectious clone (ic) expressing the spike protein of GD03T0013 (GenBank no. AY525636) (icGD03; Deming et al., 2006). Primary intranasal infection with 10³  TCID₅₀/hamster with SARS-CoV (Urbani, HK-39 or Frk-1) results in virus titers of >10⁷  TCID₅₀/g of lung by day 2 p.i. (Fig. 3A–C), whereas primary infection with icGD03 results in significantly lower titers (10⁴  TCID₅₀/g on day 2 p.i.) (Fig. 3D). Our findings suggest that compared with the Urbani strain, icGD03 is slightly attenuated in the hamster model with a 1000-fold reduction in peak virus titers in the lungs following infection, and that SARS-CoV Frk-1 may be slightly more virulent than other strains. Although dose-dependent mortality was not observed, 3 of 20 hamsters died following infection with SARS-CoV Frk-1, and peak virus titers in lungs are higher than peak virus titers following infection with Urbani or HK-39. However, the difference in peak virus titer did not achieve statistical significance (Fig. 3A–C). Histopathological findings in lungs following infection with SARS-CoV (HK-39 or Frk-1) are indistinguishable from those reported following infection with SARS-CoV (Urbani) (Fig. 4 ). The spike proteins of the SARS-CoV Urbani and HK-39 isolates are identical in amino acid sequence, and the Frk-1 spike protein sequence differs from these by only one amino acid in the S2 domain (L1148F). Antibodies detected in hamster sera following primary SARS-CoV infections cross-neutralize the various virus strains and homologous viruses to comparable degrees (Urbani, Frk-1 and HK-39: ~1:500 to 1:1000), and hamsters infected with each of these strains of SARS-CoV are protected from re-infection with the homologous virus or heterologous viruses (Fig. 3A–D).

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Fig. 3

Primary replication and replication after challenge of SARS-CoV in hamster lungs. Golden Syrian hamsters (50–55 days old) were intranasally inoculated with one of four SARS-CoV strains (A) HK-39 (N = 16), (B) Urbani (N = 16), (C) Frk-1 (N = 16), and (D) icGD03 (N = 12) of SARS-CoV (10³ TCID₅₀/hamster). For evaluating viral replication following primary (1°) infection, four hamsters per strain (three hamsters for icGD03) were sacrificed at day 2 post-infection. For evaluation of protection from secondary (2°) infection, the remaining hamsters were challenged 28 days after 1° infection with the indicated homologous or heterologous strain of SARS-CoV (N = 4 per group; or N = 3/group for icGD03; 10³ TCID₅₀/hamster) and sacrificed 2 days later. Bars indicate the mean titer of virus detected in 10% (w/v) lung homogenates by TCID₅₀ assay on Vero cells (1° = day 2 after initial infection; 2° = day 2 after challenge). Error bars indicate standard error. Dashed line indicates limit of detection, 10^1.5 TCID₅₀/g lung.

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Fig. 4

Pathology in lungs of golden Syrian hamsters following SARS-CoV infection. Lungs from SARS-CoV-infected (Urbani, A and B; Frankfurt-1, C and D; HKU-39849, E and D) hamsters at day 5 p.i. show confluent pneumonic consolidation (hematoxylin-and-eosin stain, A, C, and E), and SARS-CoV antigens (IHC assays, antigen in red; B, D, and F) in consolidated areas (asterisk) and bronchial epithelium (arrows). Original magnifications: 25× (A, C and E); 50× (B, D and F).

Although hamsters are not an inbred species, the level of genetic variability among golden Syrian hamsters is limited because they have been a closed, outbred colony since 1951. Furthermore, since hamsters demonstrate high levels of viral replication, pronounced and widespread histopathological findings in the lungs, and some clinical evidence of infection (Table 1), they are suitable for vaccine efficacy, immunoprophylaxis and treatment studies. Although the availability of immunological reagents for the hamster are limited, the use of real-time RT-PCR to study cytokine and chemokine responses following infection may reveal mechanisms of pathogenesis in this model and will lead to a better understanding of the pathogenesis of SARS in humans.

2.4. Ferrets

Ferrets (Mustela furo) support SARS-CoV replication and develop multifocal pulmonary lesions involving 5–10% of the surface area of the lung (ter Meulen et al., 2004). Ferrets infected with 10³ or 10⁴  TCID₅₀ sustain virus replication at 10⁶  TCID₅₀/mL at day 4 p.i. (Martina et al., 2003, ter Meulen et al., 2004) Two groups of investigators have utilized the ferret model with somewhat different findings (Martina et al., 2003, ter Meulen et al., 2004, Weingartl et al., 2004b), suggesting that this model needs further characterization for vaccine efficacy and immunoprophylaxis studies. Clinical symptoms following SARS-CoV infection were reported in one study (Martina et al., 2003) but not in two others (ter Meulen et al., 2004, Weingartl et al., 2004b). Additional studies would help define the biological variability of this outbred species and the potential contribution of co-pathogens to the variability observed between the different studies. Finally although the ferret model has been used successfully for immunotherapy and prophylaxis studies (ter Meulen et al., 2004), further evaluation is needed to understand the poor efficacy and immunogenicity seen in MVA-vectored, SARS-vaccinated ferrets (Weingartl et al., 2004b) compared to that observed in other animals immunized with a similar vaccine (Bisht et al., 2004, Chen et al., 2005).

2.5. Non-human primates (NHP)

NHPs support viral replication and pneumonitis with variable clinical symptoms and pathology, depending upon the species. Various strains of SARS-CoV, including Urbani, PUMC-01 and HKU-39849, have been evaluated in rhesus macaques (McAuliffe et al., 2004, Qin et al., 2005, Rowe et al., 2004), cynomolgus macaques (Fouchier et al., 2003, Kuiken et al., 2003, Lawler et al., 2006, McAuliffe et al., 2004, Qin et al., 2006, Rowe et al., 2004), common marmosets (Greenough et al., 2005b), African green monkeys (McAuliffe et al., 2004), squirrel monkeys and mustached tamarins. Squirrel monkeys and mustached tamarins were not susceptible to SARS-CoV Urbani infection (authors’ unpublished data). Virus was not detected in the respiratory tract following inoculation and monkeys did not develop antibodies to SARS-CoV. All other NHP species studied were susceptible to infection to variable degrees. Of these susceptible species, no single model is preferred to any other. Each support viral replication in lungs (Table 2 ) and viral shedding in respiratory secretions and most develop some histopathological changes consistent with human infections including infection of type I and type II pneumocytes and diffuse alveolar damage. Few vaccine candidates have been evaluated in NHPs. The vaccines that were evaluated in NHPs were efficacious in reducing titers of wild-type SARS-CoV challenge virus replication and/or histopathological changes in immunized animals compared to mock-immunized controls. Due to individual variation in responses of outbred animals, small groups of monkeys (N  = 4 or 5) are not large enough to evaluate statistical outcomes in these studies. Therefore, special considerations should be given before conducting vaccine efficacy studies in NHPs. Challenge studies should be conducted in large numbers (N  = 10–12 per group), and consideration should also be given to the history of the animals used, utilizing the highest quality of monkeys (avoiding those that have had free-range periods in life or those that are known to be infected with co-pathogens) available (Roberts et al., 2006b). In vaccine efficacy studies, immunological responses should also be evaluated. Due to the lack of available immunological reagents (including micro-array assays) for other NHP species, rhesus and cynomolgus macaques are likely to be the most informative of the non-human primate models for continued SARS research. Despite the limitations of NHP models, evaluation of vaccine candidates in NHPs may be necessary to proceed to clinical studies. The cost for large studies that will provide the needed sample size and the other special considerations mentioned will limit these vaccine efficacy studies to the most promising candidates.

3.1. Immunoprophylaxis and therapy

Prophylactically administered monoclonal antibodies specific to the SARS spike protein and passive transfer of SARS-CoV hyper-immune sera to naive mice (Greenough et al., 2005a, Subbarao et al., 2004, Sui et al., 2004, Traggiai et al., 2004, X. Wang et al., 2005), hamsters (Roberts et al., 2006a) and ferrets (ter Meulen et al., 2004) prevent or reduce SARS-CoV replication and associated disease following challenge. Monoclonal antibodies specific to the SARS spike protein administered therapeutically (i.e. after the onset of infection) prevent further increase in viral burden and reduce associated disease (e.g. consolidation) in hamsters (Roberts et al., 2006a). Sera from mice immunized with recombinant spike protein, inactivated whole SARS-CoV, live-viral vectored vaccines expressing the spike protein (VSV-S or MVA-S) or DNA vaccines (expressing SARS-S and -M proteins) have been shown to contain neutralizing antibodies to SARS-CoV (Table 3, Table 4 ). Passive transfer of these immune sera to naive mice prevents infection with SARS-CoV (Bisht et al., 2004, Kapadia et al., 2005, Stadler et al., 2005, Yang et al., 2004). Although cell-mediated immunity may have a protective role in viral clearance or resolution of disease in the human population, antibody alone has been shown to have both prophylactic and therapeutic benefits in animal models. The data from several studies strongly suggests that an effective SARS-CoV vaccine will be one that induces high and sustained levels of neutralizing antibodies and that administration of neutralizing MAbs specific to the SARS-CoV spike protein may be a useful strategy in post-exposure treatment and prophylaxis in at risk populations.

3.2. Recombinant protein and inactivated whole-virus vaccines

The safety of vaccines should always be of paramount consideration and must always be empirically determined. Recombinant protein vaccines are well accepted as being among the safest vaccines although they are not always cost-effective. Recombinant protein vaccines are excellent at eliciting sterilizing immunity that can last up to several years depending upon the immunogenicity of the protein. These vaccines do not carry the risks of incomplete inactivation, genetic recombination with circulating viruses, or reversion to virulent phenotypes. Recombinant proteins are often also very stable. Furthermore, adjuvants such as aluminum salts (Al(OH)₃ and Al₂(SO₄)₃ or alum) or a squalene emulsion (MF59) (licensed for use in the US and Europe, respectively) may be added to the formulation of protein-based vaccines to increase immunogenicity.

The SARS-CoV spike (S) protein has been identified as the major target of neutralizing antibodies (Buchholz et al., 2004, Pang et al., 2004, Sui et al., 2005, S. Wang et al., 2005, Z.Y. Yang et al., 2005, H. Zhang et al., 2004, Y. Zhang et al., 2004, Zhao et al., 2004, Zhou et al., 2004). One study demonstrated neutralizing antibodies specific for the membrane (M) protein (Pang et al., 2004). The role of antibodies specific to the SARS-S protein in conferring protection has also been well established (Greenough et al., 2005a, Roberts et al., 2006a, Subbarao et al., 2004, Sui et al., 2005, ter Meulen et al., 2004, Traggiai et al., 2004, X. Wang et al., 2005). As such, vaccines containing recombinant SARS spike proteins or neutralizing epitopes of the spike protein should be fairly attractive. In fact, spike protein and trimers of spike proteins have been expressed from baculovirus or semliki-virus-defective particle expression systems, respectively, and were evaluated for their safety, immunogenicity and efficacy in murine and hamster models (Bisht et al., 2005, Kam et al., 2007). These proteins have demonstrated excellent immunogenicity (Table 3, Table 4) and efficacy in protection from challenge in animal models.

Inactivated, whole-virus vaccines have also been shown to be immunogenic and efficacious (Table 3, Table 4). Like recombinant protein vaccines, these vaccines contain substantial amounts of the spike protein, and they elicit neutralizing antibodies at levels similar to those elicited by recombinant protein vaccines (Table 3) that confer protection from subsequent challenge in vaccinated animals. Like recombinant protein vaccines, inactivated, whole-virus vaccines would likely have a relatively stable formulation. Potential benefits of inactivated, whole-virus vaccines over recombinant protein vaccines are that the conformation of the spike protein may be maintained and that antibodies to other viral proteins may contribute to protective immunity. Major disadvantages of inactivated, whole-virus vaccines would be the need to work with whole infectious virus in the preparation of the vaccine and the potential for incomplete inactivation in the processing and manufacturing of the vaccine.

3.3. Vectored vaccines

Although vaccines that elicit both humoral and cellular immunity may provide theoretical advantages, vaccines that produce a neutralizing antibody response may be sufficient for protection from SARS-CoV infection. Convalescent human sera had a mean plaque reduction neutralization titer of 1:61 with a normal distribution of titers around the mean (J.S. Zhang et al., 2005) but the titer of neutralizing antibodies necessary to achieve protection in a human population exposed to SARS-CoV is not known. A recombinant-, DNA-, or live-vectored vaccine, or a combination of these, have the advantage that laboratory workers are not exposed to live SARS-CoV during vaccine production.

Although DNA vectors have potential to be safe, stable and relatively inexpensive vaccines, they have not been proven to be highly efficacious or immunogenic in humans. When DNA vaccines expressing the SARS-S protein were evaluated in mice, they stimulated both humoral and cell-mediated immunity (Z. Wang et al., 2005, Yang et al., 2004) and were efficacious. Antibodies to the S gene were responsible for protection (Yang et al., 2004). SARS-CoV DNA vaccines have demonstrated greater potential in prime-boost regimens in species in which robust immunity is not induced by a DNA vector alone (Table 4). However, such combinations may lessen the economic benefit of a DNA-vector approach and necessitate licensure of more than one vaccine for human use.

Several live viruses have been tested as vectors to express SARS-CoV proteins, including BHPIV3, MVA, VSV and adenovirus (Table 4). Not all vectors were equally immunogenic, and efficacy data were not available from all the studies. Nevertheless, those vectors that elicited neutralizing antibodies and protective immunity were those that expressed the spike protein of SARS-CoV. Vectors expressing other SARS-CoV proteins including M and N did not confer protection from challenge with SARS-CoV, and the addition of the M and N genes did not enhance the immunogenicity or efficacy or the vaccine encoding the spike protein alone (Buchholz et al., 2004). In senescent mice, immunization with a vectored vaccine expressing the SARS-CoV N protein alone, or with co-administered spike protein. Enhanced pathology was seen following challenge (Deming et al., 2006). A summary of the vectored vaccines tested, their immunogenicity and protective efficacy and the animal models in which they were tested are listed in Table 4. Of all the live-vectored vaccines tested to date, only one construct (MVA-S) was variably immunogenic and variably efficacious. In ferrets, vaccination with this vector did not confer protection and may have led to formation of hepatic lesions following challenge with SARS-CoV. These findings contrast with other data reported using a MVA-S-vectored vaccine in mice and rhesus macaques where they were significantly immunogenic and efficacious. It is possible that the immunogenicity of MVA-vectored vaccines, like DNA vaccines varies in different species.

4.2. Enhancement of viral entry in vitro

Pseudo-typed lentiviruses, expressing the spike protein from a SARS-CoV isolated from civet cats, have demonstrated enhanced entry into a human renal adenocarcinoma cell line 786-O when incubated with antibodies to human SARS-CoV isolates (Z.Y. Yang et al., 2005). Enhancement was not observed with pseudo-typed viruses expressing the spike protein of a human SARS-CoV isolate. Enhancement has only been demonstrated at the level of virus entry and not in virus replication, and it is yet to be determined if enhanced replication occurs in the presence of sub-neutralizing levels of antibodies. Furthermore, these in vitro findings have not been linked to any known component of human disease or infection in vivo in animal models; this phenomenon has only been reported with pseudo-typed viruses and has not been observed with human isolates of SARS-CoV; and these observations in 786-O cells were not reproducible according to He et al. (2006). Further in vitro experiments examining productive or enhanced SARS-CoV (HK-39) replication in differentiated human macrophages in the presence or absence of SARS-specific antibodies demonstrated that SARS-CoV was able to enter these cells and that entry was not enhanced by antibody to SARS-CoV. Viable virus was not recovered from these macrophage cultures two days after viral entry, and qRT-PCR analysis showed little to no viral replication in these cells (ter Meulen et al., 2006). Ex vivo experiments examining SARS-CoV (Urbani and mouse-adapted strain MA15, Roberts et al., 2007) replication in primary murine (BALB/c, BL/6 and 129S6 strains) macrophages, isolated from the lungs, spleen and peritoneum, failed to demonstrate productive infection as well (Roberts et al., unpublished data).

This research was supported in part by the Intramural Research Program of the NIH, NIAID. We acknowledge Dr. Marisa St. Claire and staff at Bioqual and Dr. Wun Ju Shieh from CDC, IDPA for participation and evaluation of SARS-CoV infection in squirrel monkeys and mustached tamarin monkeys.