Pathogenesis of SARS in senescent mice: ? We investigated the response of the immune system to SARS-CoV infection in senescent BALB/c mice by flow cytometry and found that T cells undergo phenotypic and functional changes following encounter with viral antigen and migrate to the lungs, where they develop into a highly activated/effector population. In the spleen, the changes in T cells from naive to activated/effector cell profiles suggest a systemic virus-mediated T cell activation. Thus, upon viral antigenic stimulation, T cells differentiate and acquire the capacity to migrate from secondary lymphoid tissues and traffic to the sites of infection. We identified a biphasic pattern of expression of inflammatory mediators (cytokine, chemokine and receptor) at protein and mRNA levels in the lungs of SARS-CoV-infected senescent BALB/c mice, and demonstrated that the local increase of these inflammatory mediators correlated with the migration and activation of inflammatory cells into the lungs.? In collaboration with Michael Katze, University of Washington, we compared gene expression profiles from SARS-CoV-infected young mice that show minimal disease with those from SARS-CoV-infected aged mice that develop pneumonitis to define the extent to which the host response determined the outcome of infection. We identified differences in the pattern, magnitude and duration of the host response to SARS-CoV infection in the two groups of mice. In young mice, genes related to cellular development, cell growth and cell cycle were down-regulated during peak viral replication and these transcripts returned to basal levels as virus was cleared. In contrast, older mice had a greater number of elevated immune response and cell-to-cell signaling genes, and expression of many genes was sustained even after viral clearance, suggesting an exacerbated host response to virus. Interestingly, in SARS-CoV infected aged mice, a subset of genes was induced in a biphasic pattern that correlated with peak viral replication and a subsequent influx of lymphocytes and severe histopathologic changes in the lungs discussed above. The aged mice experiencing infection with SARS-CoV show an up-regulation of an abundance of host cellular genes in response to the virus but fail to bring this response under control even as the virus is cleared while young mice are able to clear SARS-CoV rapidly and show a controlled host response. ? Evaluation of Strategies to Prevent SARS-CoV infections: ? 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. We tested two preventive strategies, vaccination and passive transfer of serum antibody, to determine the extent of protection achieved against SARS-CoV challenge in animal models. The vaccine studies demonstrated three important findings: 1) the spike glycoprotein is the major protective antigen of SARS-CoV; 2) vaccines that express the spike glycoprotein induce neutralizing antibodies; and 3) vaccines that induce a neutralizing antibody response protect animals from replication of challenge virus. ? One of the concerns in the field of SARS research is the re-emergence of SARS-CoV or a related SARS-CoV-like virus. Masked palm civets were identified as carriers of SARS-CoV and horseshoe bats carry a SARS-CoV-like virus. If SARS were to reappear from an animal reservoir, it would be more likely to resemble the animal virus rather than the prototype SARS-CoV. However, few zoonotic strains of SARS-CoV have been isolated and maintained in cell culture and this presents a challenge in assessing the efficacy of candidate SARS vaccines against zoonotic strains. Ralph Baric (UNC) has applied synthetic biology and reverse genetics techniques to generate recombinant isogenic viruses bearing variant spike glycoproteins derived from animal sources.? 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. ? We participated in a large multi-center collaboration led by Dimiter Dimitrov from NCI, to demonstrate the ability of two human antibodies that bind to a region on the receptor-binding domain of the SARS-CoV spike glycoprotein to cross-neutralize human and animal SARS-CoV isolates. Both antibodies potently cross-neutralized human and civet SARS-CoV strains in vitro. Mice that received either mAb were fully protected from infection with a human isolate of SARS-CoV and were partially protected from infection with a civet SARS-CoV-like virus. These antibodies exhibit cross-reactivity against isolates from the two SARS outbreaks in humans and isolates from palm civets, and could have potential applications for diagnosis, prophylaxis and treatment of SARS CoV infections. The ability of these human mAbs 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: ? Dr. Luis Enjuanes from Madrid generated a recombinant SARS coronavirus lacking the E gene (rSARS-CoV-E). The recombinant virus lacking the E gene (rSARS-CoV-E) 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-E virus did not cause clinical illness in hamsters as measured by use of an activity wheel and 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-E virus than with the recombinant wild-type virus. ? Based on these findings, we evaluated the immunogenicity and efficacy of this virus as a live attenuated vaccine. Hamsters immunized with rSARS-CoV-E developed high serum neutralizing antibody titers and were protected from replication of homologous and heterologous SARS-CoV in the upper and lower respiratory tract. rSARS-CoV-E -immunized hamsters remained active following wild-type virus challenge while mock-immunized hamsters displayed decreased activity. Widespread eosinophilic pulmonary infiltrates described following challenge in mice vaccinated with an alphavirus expressing the SARS-CoV N protein were not prominent in hamsters vaccinated with the rSARS-CoV-E virus. Following challenge with homologous or heterologous virus, the lungs of mock-immunized hamsters had focal intense inflammatory infiltrates and viral antigen was present. However, hamsters immunized with rSARS-CoV-E or rSARS-CoV viruses showed only mild focal infiltrates in the lungs and viral antigen was not detected. These results correlated with the activity of the hamsters and the quantitative virologic data. ? The ability of the rSARS-CoV-E vaccine to protect against challenge with the heterologous virus is significant because this virus strain is antigenically one of the most divergent strains from SARS-CoV Urbani; it clusters phylogenetically with the animal SARS-CoV isolates and was selected as a representative of an animal SARS-CoV because if SARS were to reemerge, it would likely come from an animal source. Thus, despite being attenuated in replication in the respiratory tract, rSARS-CoV-E virus has promise as a live attenuated vaccine.
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