(1) To develop animal disease (end host) and persistence (reservoir host) models: Over the past two years we have developed and characterized nonhuman primate disease models for infections with influenza A viruses, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and hantaviruses causing Hantavirus Pulmonary Syndrome (HPS). The two nonhuman primate MERS models, rhesus macaque and common marmoset, were further investigated. A comparative pathology study was performed to increase our understanding why disease in the rhesus macaque is mild-moderate, whereas disease in the common marmoset is moderate-severe. The results suggested that increased virus replication and the local immune response to MERS-CoV infection play a role in the severity of pulmonary pathology. We also investigated whether domestic pigs could be used as an alternative MERS model. Pigs were inoculated intranasally and intratracheally with a high dose of MERS-CoV. Unexpectedly, pigs did not develop signs of disease nor lesions in the respiratory tract. We have also further characterized the rhesus macaque HPS model to investigate mechanisms of disease pathogenesis. We are currently studying specific inflammatory events to gain understanding of what contributes to disease as well as to develop a therapeutic. Much of our work on hantaviruses in the past year was aimed at determining how the natural reservoirs are able to support high levels of virus replication without disease. We have previously shown that Sin Nombre virus elicits an initial inflammatory response in deer mice, the natural reservoir, but this response turned into an active anti-inflammatory response, as indicated by the activity of virus-specific T regulatory cells. Currently we are determining the requirement of these T regulatory cells to the suppression of the anti-inflammatory response by using T cell depletion strategies. (2) To identify and characterize determinants of viral pathogenicity to develop antivirals: Severe influenza virus infections are often associated with bacterial co-infections. In order to study a potentiating effect of co-infection we performed a study in cynomolgus macaques using a moderately severe pandemic H1N1 strain (Ca04) and Methicillin-resistant Staphylococcus aureus (MRSA). Animals infected with MRSA only were largely asymptomatic, whereas animals infected with Ca04 only developed moderate pulmonary disease. Interestingly, animals initially infected with MRSA followed by Ca04 showed a dramatic reduction in clinical signs, whereas those initially infected with Ca04 showed enhanced clinical disease. Similar studies were performed with a seasonal H3N2 virus and MRSA, in which we did not see disease reduction or enhancement. Studies to decipher the mechanisms behind these observations were objectives over the past year and are still ongoing. Comorbidities are frequently reported in severe MERS cases. Thus, we tested the effect of immunosuppression on outcome of MERS-CoV infection in the rhesus macaque model. Animals were immunosuppressed through treatment with cyclophosphamide and dexamethasone before MERS-CoV inoculation. Immunosuppressed macaques did not develop more severe disease than immunocompetent animals, but they shed more virus, and viral loads in the lungs were significantly higher. Despite the increased virus replication, and in line with lack of increase in clinical disease, histological examination of the lungs showed a reduced inflammatory response in immunosuppressed macaques as compared to normal animals. These results suggested that the immune response to infection plays an important role in MERS-CoV pathogenesis. A novel henipavirus, Cedar paromyxovirus (CedPV) has been isolated from pteropus bats in Australia that is closely related to Nipah virus but has not been associated with human disease. To elucidate mechanisms of disease induced by Nipah virus, we have begun to use the hamster model of henipavirus disease to characterize and compare infection and pathogenesis. CedPV replicated in hamsters, yet did not cause any conspicuous pathology. In vitro studies suggest that CedPV elicits a strong innate immune response, whereas Nipah antagonizes this response, allowing for unchecked virus replication, resulting in massive cytopathology. These studies will continue and hopeful provide new insight into henipavirus pathogenesis that can be used to develop antivirals/therapeutics. (3) To identify and characterize host responses to viral infection to develop therapeutics: For MERS, a first treatment study using a combination of ribavirin and interferon was performed in the rhesus macaque model showing efficacy as demonstrated by reduced clinical disease signs and viral load in lung tissue. In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV. The post-exposure efficacy of nebulized griffithsin in the rhesus macaque model showed moderate reduction of viral load but did not significantly reduce disease signs. We are currently refining the treatment scheme. We are also in the process of testing the efficacy of several monoclonal antibodies (mAb) as a treatment for MERS-CoV infection in the common marmoset. These mAb have shown efficacy in mouse models of MERS-CoV infection and, if shown to reduce disease burden in the common marmoset model, can quickly be used in patients. (4) To develop protective vaccines: We continued with our efforts to develop a universal vaccine against influenza A viruses. We currently are applying two approaches: i) expression of highly conserved B cell epitopes from two separate helical regions within the hemagglutinin stalk that have shown to afford heterosubtypic binding and protection, and ii) removal of hemagglutinin globular region to increase antibody responses against otherwise poorly antigenic epitopes. We used the Cytomegalovirus (CMV) vector platform for these studies, which is able to induce long-lasting immune responses (both T cell and antibody). Unfortunately, first attempts using the mouse model of influenza A viruses were rather discouraging. We will continue to optimize the CMV platform but also started to use the vesicular stomatitis virus (VSV) as an alternative platform. For MERS, we have obtained very encouraging results with a DNA vaccine platform encoding a codon-optimized consensus spike protein. This vaccine candidate induced potent cellular immunity and antigen specific neutralizing antibodies in three animal species, mice, macaques and camels. Vaccinated macaques were protected against MERS-CoV challenge and did not show any clinical or radiographic signs of pneumonia. The vaccine candidate will be further tested for its use in emergency situations to prevent MERS-CoV infection. In an effort to generate an effective countermeasure for Nipah virus infection, we used the VSV based vaccine approach to express one of the glycoproteins of Nipah virus as an immunogen. These vaccines elicited strong antibody responses in hamsters and protected them from lethal, high dose Nipah virus challenge. We also showed efficacy of one of these VSV vaccines in the African green monkey model. Furthermore, we were able to characterize the T cell response to vaccination and challenge and showed that not only is a strong neutralizing response elicited, but the vaccine primes the CD8+ T cell response as well. To investigate the limits of the efficacy of this vaccine, we used the hamster model and showed that this vaccine provides at least partial protection when given as late as on the day of virus challenge. Future work will examine the mechanisms of protection and test the vaccine's peri-exposure efficacy in nonhuman primates.
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