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Principal Investigator: Emmie De Wit
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2022
Award: $1,400,690
Funding agency: National Institute of Allergy and Infectious Diseases
In any given year, lower respiratory tract infections are the leading cause of infectious disease deaths worldwide, and the fifth most important cause of death overall. Respiratory viruses keep emerging at a steady pace (e.g. MERS-CoV, enterovirus D68, avian influenza viruses), adding to the burden of respiratory tract infections on global health. In 2019-2022, the emergence of SARS-CoV-2 and the resulting COVID-19 pandemic highlighted how devastating the effect of emerging respiratory viruses on global public health and economies can be. The COVID-19 pandemic also showed once again the difficulty of effectively treating severe viral lower respiratory tract infections. Major advances have been made in our knowledge of the pathogenic processes involved in severe respiratory disease over the past decade; however, few successful treatments have made their way into the clinic. Although many clinical trials have been performed and are ongoing in COVID-19 patients, very few of those have generated promising results. Thus, it is clear that our current understanding of the pathogenesis of viral lower respiratory tract infections is insufficient to drive the development of effective treatments. The main goal of the Molecular Pathogenesis Unit is to contribute to our understanding of the pathogenesis of such as Nipah virus, influenza A virus and coronaviruses on the level of the host and individual cell. Our main focus is currently on the respiratory disease these viruses cause, but we are expanding our research into the neurological complications caused by these viruses. Ultimately, our work will lead to the identification of common pathways involved in lower respiratory tract and neurological disease progression and druggable targets within those pathways.
Our research efforts in FY2022 were divided between responding to the COVID-19 pandemic and developing new tools for our pathogenesis studies.
Our pandemic response work consisted of supporting several clinical studies, studying pathogenesis, and testing of countermeasures. We studied the pathogenicity of variants of concern (VOC) Alpha and Beta in rhesus macaques, and Delta and Omicron in human alveolar organoids. In rhesus macaques, we showed differences in pathogenicity between the VOC in replication kinetics, disease severity, and inflammatory response. The Beta VOC was less pathogenic than the ancestral D614G virus, while the Alpha VOC was equally pathogenic, but caused a more pronounced inflammatory response.
We also studied the role of age as a risk factor for severe COVID-19 in the rhesus macaque model since advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we inoculated eight older and eight younger macaques with SARS-CoV-2. Differences in clinical signs and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. Thus, age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.
Unfortunately, remdesivir has had limited utility in COVID-19 patients because of the necessity to administer remdesivir intravenously, which has generally limited its use to hospitalized patients, where remdesivir has a limited benefit. To address this issue, Gilead Sciences developed a new formulation of remdesivir that can be administered subcutaneously, enabling outpatient administration and reducing the time from diagnosis to treatment. Our laboratory tested this novel formulation in the rhesus macaque model of SARS-CoV-2 infection. Compared to vehicle-treated animals, macaques treated with subcutaneous remdesivir from 12 hours through 6 days post inoculation showed reduced signs of respiratory disease, a reduction of virus replication in the lower respiratory tract, and an absence of interstitial pneumonia. Thus, early subcutaneous administration of remdesivir can protect from lower respiratory tract disease caused by SARS-CoV-2. This work has implications not only for the treatment of SARS-CoV-2 infection, but for other emerging viruses as well, e.g. henipaviruses and filoviruses, against which intravenous remdesivir treatment also has proven efficacy in vivo.
We continued to develop human alveolar organoids in our laboratory. We optimized the methods to establish these organoids from human lung tissue, testing different protocols to enrich for epithelial cells, such as MACS and flow cytometry, established essays to test cell viability, and optimized infection and sample collection protocols for SARS-CoV-2 and influenza A virus. We extensively characterized the cellular components of our alveolar organoids using electron microscopy, fluorescence microscopy, qRT-PCR profiling, and histology. We found that the human alveolar organoids express the expected epithelial marker EpCam, as well as type II pneumocyte markers, such as HTII-280 and prosurfactant protein C. The cells also express the SARS-CoV-2 receptor ACE2. We performed a study t compare SARS-CoV-2 VOC Delta and Omicron to the ancestral D614G virus and found that the Delta VOC replicated most efficiently, and Omicron replicated in organoids derived from only 1 of 4 human donors. Since these data correlate with pathogenicity data from human cases and animal models, we will expand the work using these organoids to study specific aspects of viral respiratory tract disease, such as breakdown of the epithelial barrier function that is a major cause of acute respiratory distress, as well as the host response to infection in the most relevant, functional cell types and in a host that can be rarely studied in sufficient detail, humans.
MPUs viruses of interest, SARS-CoV-2, Nipah virus and 1918 H1N1 influenza virus can all cause neurological complications. Whereas neurological complication occurred in a small subset of patients with COVID-19 or the Spanish flu, Nipah virus neurological complications contribute equally to the lethality of this virus in humans. Although the majority of Nipah virus infections is fatal, 60% of the survivors suffer from long term neurologic sequelae and late onset and relapsed encephalitis have been observed even more than 10 years after the initial infection. There is a paucity of data from human NiV cases and Nipah virus animal experiments have mostly been skewed towards models with fatal respiratory disease without neurological complications. Thus, the pathogenesis of NiV neurological disease during the acute, convalescent and relapse phase is poorly understood. We are trying to fill that gap in our understanding in two ways: by studying archived tissue samples from animal experiments and by developing a model for Nipah virus neuropathogenesis in hamsters. Using histologic characterization of African green monkeys infected with Nipah virus, we have shown which cell types are infected, which cell types mount an inflammatory response to infection, and which cells are extravasating into the CNS during infection. We have started to develop a new Syrian hamster model of neurological disease using a combination of Nipah virus inoculation and suboptimal remdesivir treatment to skew the disease in these animals to neurological signs rather than respiratory disease. Preliminary data show that this approach might be feasible, although we need to optimize the model further.
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