Multiplexed proteomics and imaging of resolving and lethal SARS-CoV-2 infection in the lung (preprint)

Normal tissue physiology and repair depends on communication with the immune system. Understanding this communication at the molecular level in intact tissue requires new methods. The consequences of SARS-CoV-2 infection, which can result in acute respiratory distress, thrombosis and death, has been studied primarily in accessible liquid specimens such as blood, sputum and bronchoalveolar lavage, all of which are peripheral to the primary site of infection in the lung. Here, we describe the combined use of multiplexed deep proteomics with multiplexed imaging to profile infection and its sequelae directly in fixed lung tissue specimens obtained from necropsy of infected animals and autopsy of human decedents. We characterize multiple steps in disease response from cytokine accumulation and protein phosphorylation to activation of receptors, changes in signaling pathways, and crosslinking of fibrin to form clots. Our data reveal significant differences between naturally resolving SARS-CoV-2 infection in rhesus macaques and lethal COVID-19 in humans. The approach we describe is broadly applicable to other tissues and diseases.

Evidence of antigenic imprinting in sequential Sarbecovirus immunization (preprint)

Antigenic imprinting, which describes the bias of antibody response due to previous immune history, can influence vaccine effectiveness and has been reported in different viruses. Give that COVID-19 vaccine development is currently a major focus of the world, there is a lack of understanding of how background immunity influence antibody response to SARS-CoV-2. This study provides evidence for antigenic imprinting in Sarbecovirus, which is the subgenus that SARS-CoV-2 belongs to. Specifically, we sequentially immunized mice with two antigenically distinct Sarbecovirus strains, namely SARS-CoV and SARS-CoV-2. We found that the neutralizing antibodies triggered by the sequentially immunization are dominantly against the one that is used for priming. Given that the impact of the background immunity on COVID-19 is still unclear, our results will provide important insights into the pathogenesis of this disease as well as COVID-19 vaccination strategy.

The Strand-biased Transcription of SARS-CoV-2 and Unbalanced Inhibition by Remdesivir (preprint)

SARS-CoV-2, a positive single-stranded RNA virus, caused the COVID-19 pandemic. Although its sense-mRNA architecture was reported, its anti-sense strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand. During negative strand synthesis, apart from canonical sub-genomic ORFs, numerous non-canonical fusion transcripts are formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor Remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the anti-viral drug design.

Analysis of SteraMist ionized hydrogen peroxide technology as a method for sterilizing N95 respirators and other personal protective equipment (preprint)

ObjectiveThe COVID-19 pandemic has led to widespread shortages of personal protective equipment (PPE) for healthcare workers, including filtering facepiece respirators (FFRs) such as N95 masks. These masks are normally intended for single use, but their sterilization and subsequent reuse could substantially mitigate a world-wide shortage. DesignQuality assurance. SettingA sealed environment chamber installed in the animal facility of an academic medical center. InterventionsOne to five sterilization cycles using ionized hydrogen peroxide (iHP), generated by SteraMist(R) equipment (TOMI; Frederick, MD). Main outcome measuresPersonal protective equipment, including five N95 mask models from three manufacturers, were evaluated for efficacy of sterilization following iHP treatment (measured with bacterial spores in standard biological indicator assemblies). Additionally, N95 masks were assessed for their ability to efficiently filter particles down to 0.3{micro}m and for their ability to form an airtight seal using a quantitative fit test. Filtration efficiency was measured using ambient particulate matter at a university lab and an aerosolized NaCl challenge at a National Institute for Occupational Safety and Health (NIOSH) pre-certification laboratory. ResultsThe data demonstrate that N95 masks sterilized using SteraMist iHP technology retain function up to five cycles, the maximum number tested to date. Some but not all PPE could also be sterilized using an iHP environmental chamber, but pre-treatment with a handheld iHP generator was required for semi-enclosed surfaces such as respirator hoses. ConclusionsA typical iHP environment chamber with a volume of ~80 m3 can treat ~7000 masks per day, as well as other items of PPE, making this an effective approach for a busy medical center.

Rapid prototyping and clinical testing of a reusable face shield for health care workers responding to the COVID-19 pandemic (preprint)

Due to supply chain disruption, the COVID-19 pandemic has caused severe shortages in personal protective equipment (PPE) for health care professionals. Local fabrication based on 3D printing is one way to address this challenge, particularly in the case of simple products such as protective face shields. As a consequence, many public domain designs for face shields have become available. No clear path exists, however, for introducing a locally fabricated and unapproved product into a clinical setting. In a US health care setting, face shields are regulated by the Food and Drug Administration (FDA); similar policies exist in other countries. We describe a research protocol under which rapid iteration on an existing design, coupled with clinical feedback and real-world testing in an emergency department, allowed a face shield to be adopted by the incident command team at a major academic medical center. We describe our design and testing process and provide an overview of regulatory considerations associated with fabrication and testing of face shields and related products. All designs, materials used, testing protocols, and survey results are reported in full to facilitate the execution of similar face shield efforts in other clinical settings. Our work serves as a case study for development of a robust local response to pandemics and other health care emergencies, with implications for healthcare professionals, hospital administrators, regulatory agencies and concerned citizens.