SARS-CoV-2 pathogenesis and countermeasures

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

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 2019-2020, 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 highlighted 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. Even those therapeutics tested with positive outcomes, showed benefits only in a fraction of patients. 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. In the past year, our research has mainly focused on four aspects of COVID-19: model development, pathogenesis, support of NIH clinical work, and efficacy of potential antiviral treatments. In terms of model development, we have successfully established 3D small airway and alveolar organoids from 4 human donors. We have characterized the cellular make-up of these organoids and shown that they express markers typical of type II pneumocytes, and the receptor for SARS-CoV-2, ACE2. We have tested and optimized procedures for infection of the 3D organoids with SARS-CoV-2 and testing their viability during virus infection. We compared the ability of B.1.1.529 (Omicron) to replicate in human lung epithelium, cause cell death and induce pro-inflammatory cytokines to that of B.1.629 (Delta) and D614G (ancestral clade B.1 virus). B.1.1.529 was severely attenuated in the alveolar organoids compared to B.1.629 and D614G, and was only able to replicate to low titers in one of four donors. This is consistent with early reports of reduced B.1.1.529 replication in the lower respiratory tract of rodents, and a milder clinical course of disease in humans. In contrast, B.1.629 consistently replicated faster and to higher titers compared to B.1.1.529 and D614G in all donors tested. Despite high levels of virus replication, we did not observe significant host cell death in organoids infected with any variant. We are currently comparing the production of cytokines in the organoids infected with the different isolates and analyzing the host response to infection using transcriptomics, hoping to identify pathways underlying the differences in replicative ability between variants and hosts. Our pathogenesis studies focused on the pathogenicity of Variants of Concern and why increased age is a major risk factor for developing severe COVID-19. SARS-CoV-2 variants B.1.1.7 (Alpha) and B.1.351 (Beta) were both classified as Variant of Concern (VOC), based on their rapid spread in the population and reports on increased hospitalization rates. To compare the pathogenicity and virus shedding of VOC B.1.1.7 and B.1.351, we inoculated three groups of six rhesus macaques intranasally and intratracheally with three different SARS-CoV-2 isolates: D614G, a B.1.1.7 isolate, and a B.1.351 isolate. The B.1.1.7 VOC behaved similarly to the D614G with respect to clinical disease, virus shedding and virus replication in the respiratory tract. Inoculation with the B.1.351 isolate resulted in lower clinical scores in rhesus macaques that correlated with lower virus titers in the lungs, less severe histologic lung lesions and less viral antigen detected in the lungs. In bronchoalveolar lavages, cytokines and chemokines were upregulated on day 4 in animals inoculated with D614G and B.1.1.7 but not in those inoculated with B.1.351. In nasal samples, we did not detect upregulation of cytokines and chemokines in D614G or B.1.351-inoculated animals. However, cytokines and chemokines were upregulated in the noses of B.1.1.7-inoculated animals. To investigate the effect of age on the pathogenesis of SARS-CoV-2 infection, we inoculated eight aged (16-23 years) and eight subadult (3-5 years) rhesus macaques with SARS-CoV-2. We then used a multi-omics approach to gain a complete picture of the host response to SARS-CoV-2 in the lungs and systemically. Although older animals displayed slightly elevated clinical scores over the course of infection and recovery was somewhat slower, differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between the two age groups. Despite this limited effect of age on disease outcome, we found several striking differences in the response to SARS-CoV-2 infection through immunological and transcriptional profiling. During the acute phase of SARS-CoV-2 infection in the older animals, the local innate immune response was upregulated in both immune and non-immune cell populations through 7 dpi. In contrast, the younger animals appeared to better control the innate inflammatory response in the lungs, as evidenced by the more attenuated induction of innate pathways that occurred only in immune cells, and resolving it by 7 dpi. During the post-acute phase of the disease, the two groups diverged further, with older animals exhibiting a prolonged pro-inflammatory circulating response, as well as a delay in the activation and/or differentiation of T-cells isolated from the lungs. Concomitant with these changes within the T-cell compartment, the younger animals displayed a pro-resolving circulating milieu of lipid mediators and cytokines at later timepoints, while the older animals did not. We supported several clinical studies with our standardized SARS-CoV-2 assays. We helped the NIH Clinical Center to answer questions about the use of SARS-CoV-2 diagnostics for return-to-work testing early in the pandemic, as well as helping to compare the reliability of saliva swabs versus nasopharyngeal swabs for diagnostic purposes. We also supported studies showing long-term shedding of infectious SARS-CoV-2 virus in immunocompromised patients. Finally, we completed a study assessing the efficacy of subcutaneous remdesivir treatment, as a potential replacement for intravenous administration. Two groups of six rhesus macaques were inoculated with SARS-CoV-2 and treated according to the same dosing schedule as used for intravenous remdesivir. Pharmacokinetic analyses of remdesivir and its main metabolites in the rhesus macaques indicated that the release of remdesivir is somewhat slower with subcutaneous administration, but that plasma levels persist longer. Additionally, the active triphosphate metabolite concentration in the lungs was similar with subcutaneous and intravenous administration. 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.