Animal models for SARS: ? We have studied the replication of SARS-CoV in mice, hamsters and non-human primates and established that intranasally administered SARS-CoV replicated efficiently in the respiratory tissues. Although viral antigen and nucleic acid are present in the lung tissues, this is accompanied by only mild inflammation in young mice. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, and they can be used for vaccine and immunoprophylaxis studies but immune senescence complicates pathogenesis studies. ? In order to develop a model of SARS associated with clinical illness in young mice, we serial passaged the SARS-CoV (Urbani) strain in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites, accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with the mouse-adapted virus die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The mouse-adapted virus has six coding mutations associated with adaptation and increased virulence. Intranasal inoculation with the mouse-adapted virus reproduces many aspects of disease seen in severe human cases of SARS and will enhance the use of the mouse model for SARS because infection with this virus causes morbidity, mortality, and pulmonary pathology. This virus will be of value in evaluation of the efficacy of vaccines and antivirals.? The golden Syrian hamster is an excellent model for SARS-CoV infection because viral replication is accompanied by pathological changes in the lungs including pneumonitis and consolidation. Although histopathological signs of disease are pronounced, clinical symptoms of disease have been difficult to identify in hamsters. We used exercise wheels to evaluate overnight activity and we now have objective evidence of clinical illness in hamsters. 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. Antibodies detected in hamster sera following primary SARS-CoV infection with three different strains cross-neutralize the homologous and heterologous viruses to comparable degrees and hamsters infected with each of these strains of SARS-CoV are protected from re-infection with the homologous virus or heterologous viruses. This observation is important because it validates the utility of the hamster model for different strains of SARS-CoV and it provides proof of concept of cross-protection among these strains.? Evaluation of Strategies to Prevent SARS-CoV infections: ? SARS coronavirus (SARS CoV) caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003-2004 by an independent animal to human transmission. We have collaborated with scientists at the NIH, at academic institutions and in industry to evaluate the immunogenicity and efficacy of a number of vaccines against SARS-CoV in animal models, The latter model supports a high level of viral replication in association with clinical illness and disease that mimics SARS in the elderly. We tested two preventive strategies, vaccination and passive transfer of serum antibody, to determine the extent of protection achieved against SARS-CoV challenge in this model. These approaches were able to achieve or induce antibody titers sufficient to reduce viral load, protect from weight loss, and reduce or eliminate histopathologic changes in the lungs of aged mice, thus validating the utility of the aged BALB/c mouse model for evaluation of the efficacy of vaccines and immunoprophylaxis. ? 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. ? ? Immunoprophylaxis studies: ? Prophylactically administered monoclonal antibodies specific to the SARS spike protein and passive transfer of SARS-CoV hyper-immune sera to nave mice, hamsters and ferrets prevent or reduce SARS-CoV replication and associated disease following challenge. We demonstrated that 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. ? The GD03 strain, which was isolated from an index patient of the second outbreak in the winter of 2003-2004, was reported to resist neutralization by some human monoclonal antibodies (hmAbs) that could potently neutralize isolates from the first outbreak in late 2002/early 2003. We demonstrated that two human monoclonal antibodies m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani, or recombinant viruses bearing the GD03 and SZ16 S glycoproteins. Both antibodies competed with the SARS CoV receptor, ACE2, for binding to the receptor binding domain of the spike protein, suggesting a mechanism of neutralization that involves interference with the SARS CoV-ACE2 interaction. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS CoV isolates with known sequences, except strains derived from bats. These antibodies are the first identified hmAbs, which exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets, and could have potential applications for diagnosis, prophylaxis and treatment of SARS CoV infections. The ability of these hmAbs to neutralize zoonotic isolates is important because if SARS-CoV reappears in nature, it will likely result from re-introduction from an animal host.? Vaccine studies: ? We have evaluated a deletion mutant of SARS-CoV that was engineered by deleting the structural E gene in an infectious cDNA clone that was generated by Dr. Luis Enjuanes. The recombinant virus lacking the E gene (rSARS-CoV-deltaE) grew in Vero E6, Huh-7 and CaCo-2 cells to titers 20, 200 and 200-fold lower than the recombinant wild-type virus, respectively, indicating that E protein was not essential for virus replication. The rSARS-CoV-deltaE virus replicated to titers 100 to 1000-fold lower than the recombinant wild-type virus in the upper and lower respiratory tract of hamsters, and the lower viral load was accompanied by less inflammation in the lungs of hamsters infected with rSARS-CoV-deltaE virus than with the recombinant wild-type virus. The utility of rSARS-CoV-deltaE that lacks the E gene as a live attenuated SARS-CoV vaccine is under evaluation.
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