Severe Acute Respiratory Syndrome (SARS) is a severe respiratory illness caused by a newly identified virus named SARS coronavirus (SARS-CoV). The disease emerged in late 2002 and spread to several countries in early 2003 and was responsible for 8,098 cases including 774 deaths worldwide. The syndrome is characterized by fever, chills or rigors, headache and non-specific symptoms such as malaise and myalgias, followed by cough, and dyspnea. The severe morbidity and mortality associated with SARS make it imperative that effective means to prevent and treat the disease be developed and evaluated, especially since it is not known whether the virus will reappear or whether it will be independently reintroduced into the human population. We have developed animal models for SARS in order to evaluate immunoprophylaxis and immunotherapy for SARS and to understand the pathogenesis of the disease. 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. Pulmonary virus replication in mice was not accompanied by clinical illness in young mice, hamsters, rhesus, cynomolgus or African green monkeys. Although viral antigen and nucleic acid are present in the lung tissues, this is accompanied by mild inflammation in young mice. In contrast, interstitial pneumonitis is seen in African green monkeys and hamsters and consolidation is seen in hamster lungs. All the animal models tested developed a serum neutralizing antibody response and were protected from re-infection 28 days following primary infection. There was no evidence of enhanced disease upon challenge in previously infected mice, hamsters or African green monkeys. SARS-CoV replicates in the respiratory tract of BALB/c mice and hamsters to levels that will permit an evaluation of the efficacy of vaccines, immunotherapeutic and antiviral drug treatment strategies. Our observations in mice and hamsters, that primary infection provides protection from re-infection and that antibody alone can protect against viral replication, suggest that vaccines that induce neutralizing antibodies and strategies for immunoprophylaxis or, perhaps, immunotherapy are likely to be effective in SARS. Pathogenesis studies: Advanced age has been repeatedly identified as an independent correlate of adverse outcome and a predictor of mortality in cases of SARS. SARS-associated mortality may exceed 50% in persons aged 60 years or older. Heightened susceptibility of the elderly to severe SARS and the ability of SARS-coronavirus to replicate in mice led us to examine whether aged mice might be susceptible to disease. SARS-CoV- infected aged mice demonstrated signs of clinical illness characterized by significant weight loss, hunching, ruffled fur and slight dehydration, signs that resolved by day 7 post-infection and mortality was not observed. Virus was detected in lungs at high titers as early as day 2 post-infection, and titers remained high on day 5 post-infection. Virus was also recovered from the upper respiratory tract and liver. Early after infection SARS-CoV antigens were detected in ciliated columnar epithelial cells of the nasal turbinates and bronchioles and in alveolar pneumocytes. Changes indicative of alveolar damage, including multifocal, interstitial infiltrates, proteinaceous deposits around alveolar walls, and intraalveolar edema, were seen beginning on day 5 post-infection. At day 9, perivascular infiltrates persisted, and the changes associated with alveolar damage were accompanied by proliferation of fibroblasts in inflammatory foci. It is possible that these foci in SARS-CoV-infected mice represent histopathologic correlates of fibrosis or scarring identified by high-resolution CT scanning of the lungs of some human patients who have recovered from severe cases of SARS. SARS CoV-infected aged mice mounted an adaptive immune response to SARS CoV infection. However, in contrast to young BALB/c and B6 mice, IFN-a, IFN-g, and TNF-a were elevated early in infection in SARS-infected, aged BALB/c mice. This suggests that a proinflammatory cytokine response may be responsible for subsequent disease associated events. In summary, we found that viral replication in aged mice was associated with clinical illness and pneumonia, demonstrating an age-related susceptibility to SARS disease in animals that parallels the human experience. Vaccine studies: 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 including inactivated, subunit, vectored, and DNA vaccines. In collaboration with Dr. Bernard Moss? laboratory (Laboratory of Viral Diseases, NIAID), we demonstrated that a secreted, glycosylated polypeptide containing amino acids 14 to 762 of the SARS-CoV spike protein expressed in recombinant baculovirus-infected insect cells administered with adjuvants saponin (QS21) or Ribi (MPL + TDM) induces protective immunity against subsequent SARS-CoV challenge in mice. A. Both regimens induced binding and neutralizing antibodies and protection against SARS-CoV intranasal infection. In collaboration with Chiron Inc., we showed that an inactivated vaccine encoding the spike glycoprotein of the SARS-CoV induces neutralizing antibody responses and protective immunity, in a mouse model. We have also collaborated with John Rose (Yale University) and Matthias Schnell (Thomas Jefferson University) to demonstrate that SARS-CoV spike protein expressed in a live rhabdovirus vectors was immunogenic in mice. Mice vaccinated with an attenuated VSV recombinant (VSV-S) expressing the SARS-CoV spike (S) protein developed SARS-neutralizing antibody and were able to control a challenge with SARS-CoV performed at 1 month or 4 months after a single vaccination. We also demonstrated that the antibody response induced by the VSV-S vaccine was sufficient for controlling SARS-CoV infection. In the case of the attenuated Rabies virus vaccine, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the rabies virus glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein, but not the N-protein, induced a strong SARS-CoV-neutralizing antibody response. Immunoprophylaxis with Monoclonal Antibodies: We extended our observation that antibodies alone were able to protect mice from SARS infection by demonstrating the ability of monoclonal antibodies (MAbs) against the spike protein of SARS-CoV to protect mice from infection with SARS-CoV. In collaboration with Dr. Donna Ambrosino at the University of Massachusetts, we evaluated MAbs derived from transgenic mice with human immunoglobulin genes that were immunized with the recombinant spike glycoprotein ectodomain of SARS-CoV. MAb-201 is a SARS-CoV specific MAb that prevents establishment of viral replication in vitro and prevents viral replication in vivo in mice, when administered prophylactically. The efficacy of MAb-201 for treatment of SARS was evaluated in golden Syrian hamsters. Post-exposure treatment with MAb-201 alleviated virus burden and the degree of associated pathology, including interstitial pneumonitis and consolidation Successful post-exposure therapy with MAb-201 in the hamster model that demonstrates viral replication and associated pulmonary pathology suggests that this MAb may be useful in the arsenal of tools to combat SARS.
Showing the most recent 10 out of 27 publications
