ABSTRACT
SARS-CoV-2 remains a significant public health threat, causing severe respiratory illness in susceptible individuals. Several effective Covid-19 vaccines have been developed but novel SARS-CoV-2 variants continuously emerge that are more transmissible and have potential to evade vaccine immune responses. We are developing a novel therapy that does not depend on an immune response, based on siRNA-mediated silencing of Angiotensin-converting enzyme 2 (ACE2) receptor and Transmembrane Serine Protease 2 (TMPRSS2). SARS-CoV-2 requires these host proteins to enter respiratory epithelial cells at the cell surface, through binding and priming of its Spike protein. As a cell model for SARS-CoV-2 infection, we have utilised primary nasal epithelial cells (NHNE), as well as HEK293T cells overexpressing ACE2 and TMPRSS2. siRNA transfection in NHNE cells led to a 78%-88% knockdown of ACE2 and TMPRSS2, as determined by qRT-PCR and western blot data. TMPRSS2 knockdown in the overexpressing HEK293T cells resulted in an 87% reduction in infectivity from SARS-CoV-2 Spike-pseudotyped lentiviruses expressing a luciferase transgene, indicative of a significant reduction in virus entry (p < 0.0001 by one-way ANOVA). We are now working to confirm these results with live SARS-CoV-2 and to test lipid nanoparticle delivery of the siRNAs to air-liquid interface grown NHNEs to more accurately model the respiratory airway. This siRNA approach could provide a novel therapy for immunocompromised individuals who do not gain sufficient protection from SARS-CoV-2 vaccines. Additionally, by targeting host proteins rather than virus components, our therapy is likely to remain effective in spite of emerging SARS-CoV-2 variants that circumvent pre-existing immune responses.
ABSTRACT
Background: In coronavirus disease 2019 (COVID-19) the need for intervention increases with disease severity and a risk prediction model that incorporates biomarkers would be beneficial for identifying patients for treatment escalation. Aim(s): To investigate biomarkers changes associated with disease severity and outcomes (mortality, thrombosis). Method(s): COVID-19 patients were sampled between April 15 and May 31 2020. Disease severity was assessed by World Health Organization (WHO) ordinal scale. 132 systemic biomarkers were investigated by routine and multiplex assays and statistical analysis performed to characterise the biomarker profile of COVID-19 patients associated with disease severity, duration, survival and thrombosis. Result(s): The study enrolled 150 COVID-19 positive adults and 16 healthy volunteers. The average age was 64 years, 59% were male, 85% had co-morbidities, 33% had a thrombotic event, and 13% died. A cross comparative analysis of biomarkers identified 13 biomarkers common to severity, mortality and thrombosis with significant correlation;including endothelial dysfunction (VWF, tPA, TFPI), hypercatabolism (low albumin, Hb, FXIII) and inflammatory response (IL-8, Osteopontin). Similarly, 14 biomarkers associated with severity and mortality included pro-inflammatory cytokines and their receptors (sTNFRII, STNFRI, sIL2a, IL6, MIP1a), neutrophils (elevated WBC, Neutrophils, TIMP1) and tissue remodelling (SCGF, EG3A). Nine biomarkers common across severity and thrombosis were angiogenesis (VEGF, LYVE1, Follistatin), acute phase response (SAP, AGP) and clot formation (Fibrinogen and PAPs). Conclusion(s): The biomarker profile associated with poorer outcomes indicates an inflammatory response, endothelial cell disruption, hypercoagulability and hypercatabolism. This study has identified several biomarkers that may be useful indicators of disease severity and progression. Further work is needed to determine how these may be used to direct clinical management. (Figure Presented).
ABSTRACT
Case Diagnosis: Rare Diagnosis of Para-Infectious SARS-COV2 Associated Acute Transverse Myelitis Based Off Clinical Presentation and CSF Studies with Negative Imaging Case Description or Program Description: Patient was admitted for two weeks of ascending paresthesias and weakness of his lower extremities around ten days after recovering from a mild SARS-COV2 associated illness. While in the hospital, his weakness progressed to flaccid paralysis of his lower extremities with a sensory level at T10. Initial workup including magnetic resonance imaging (MRI) of his brain, cervical, thoracic and lumbar spine were negative. Initial electrodiagnostic (EMG) testing was unrevealing. He received a course of intravenous immunoglobulins followed by a five day course of intravenous solumedrol, both of which did not result in any improvement. Multiple weeks into admission, the patient began to exhibit hand intrinsic weakness and paresthesias, so plasmapheresis was attempted without any change in his symptoms. Setting(s): Major Academic and Referral Center with Level 1 Adult Trauma Assessment/Results: After admission to our inpatient rehabilitation unit, repeat MRIs of his cervical and thoracic spine were again unrevealing. Initial cerebrospinal fluid analysis showed lymphocytic pleocytosis, elevated protein and positive oligoclonal bands (2). Repeat EMG obtained after his upper extremity symptoms began did not reveal a cause for his weakness. Inflammatory and neoplastic workups were negative. He also developed upper motor neuron signs on neurological examination late in his admission. Discussion (relevance): There are documented cases of neurologic complications, specifically transverse myelitis, associated with the multi-systemic inflammatory/ immunological response in the post-infectious period of SARS-COV2. What is exceedingly rare, but documented, is a diagnosis based on clinical presentation, elevated CSF protein and lymphocytes alone with the exclusion of other diagnoses. Conclusion(s): The neurologic complications of SARSCOV2 in the post-infectious period include transverse myelitis and in rare cases like this, can present without the typical findings seen on imaging of the spinal cord.