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. SARS-CoV was transmitted to humans from an animal host. Civet cats in wet markets and Chinese horseshoe bats in the wild are infected with closely related viruses. Although another epidemic of SARS has not occurred since 2003, the virus is still present in animal(s) in nature. An infected animal reservoir can be a source for the re-introduction of SARS-CoV into humans. The severe morbidity and mortality associated with SARS in 2003 make it imperative that effective means to prevent and treat the disease be developed and evaluated. 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 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 only mild inflammation in young mice. More extensive interstitial pneumonitis is seen in African green monkeys and hamsters and consolidation occurs in hamster lungs. Each of the animals tested developed a serum neutralizing antibody response and was 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 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, indicate 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 that resolved by day 7 post-infection. Virus was detected in lungs at high titers from day 2 to day 5 post-infection. 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. SARS CoV-infected aged mice mounted an adaptive immune response to SARS CoV infection. However, in contrast to young BALB/c and B6 mice, a proinflammatory cytokine response was seen early in infection in SARS-infected, aged BALB/c mice. 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. The nature of the immune cells in the lungs of SARS-infected, aged BALB/c mice is being characterized. 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 investigators in Hong Kong, we evaluated a recombinant native full-length S-protein trimer (triSpike) of SARS-CoV and analyzed the capacity of antibodies elicited by this vaccine in animals to mediate antibody-dependent enhancement of virus entry in vitro and enhancement of replication in vivo. The vaccine was able to elicit a neutralizing and protective immune response in animals. SARS-CoV-specific IgG were readily detected in sera and nasal lavages and IgG and IgA could be detected in stool samples of triSpike vaccinated mice or hamsters. Antibodies in sera and stool were capable of neutralizing SARS-CoV infection of susceptible cell lines in vitro. Sera from hamsters and mice immunized with triSpike facilitated entry of SARS-CoV pseudotyped virus into human B cell lines. Similar enhanced entry of a pseudotyped virus into human B cell lines was also detected with sera from convalescent SARS patients. Entry into human B cells occurred in a FcgRII-dependent and ACE2-independent fashion indicating that antibody-dependent enhancement of virus entry (ADE) is a novel cell entry mechanism of SARS-CoV. However, these experiments have not been performed with SARS-CoV and therefore, evidence of virus replication in B cells is lacking. Enhanced replication is characteristic of classical antibody-mediated enhancement seen in Dengue virus infection. The capacity of vaccine-induced anti-S antibodies to protect from infection in vivo was assessed using the hamster model. Vaccinated animals showed no signs of enhanced lung pathology or hepatitis and viral load was undetectable or greatly reduced in lungs following challenge with SARS-CoV. Thus, the in vitro observation of antibody enhanced entry was not seen in vivo at levels of antibody that neutralize infectivity of SARS-CoV for ACE-2 bearing cells. Our results indicate that a recombinant trimeric S protein was able to elicit an efficacious protective immune response in vivo but, if the observation of enhanced entry into B cells in vitro is shown to have an in vivo correlate, this would warrant concern in the safety evaluation of a human vaccine against SARS-CoV.
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