The wide spectrum of neuropsychiatric complications in Covid-19 patients within a multidisciplinary hospital context (preprint)

Objective: To describe the spectrum of neurological and psychiatric complications in patients with Covid-19 seen in a multidisciplinary center over six months. Methods: We conducted a retrospective, observational study on all patients showing neurological or psychiatric symptoms in the context of Covid-19 seen in the Department of Neurology and Psychiatry of the APHP-Sorbonne University. We collected demographic data, medical and treatment history, comorbidities, symptoms, date of onset, and severity of Covid-19 infection, neurological and psychiatric symptoms, neurological and psychiatric examination data and, when available, results from cerebrospinal fluid (CSF) analysis, brain magnetic resonance (MRI) imaging, 18-fluorodesoxyglucose-position emission computed tomography (FDG-PET/CT)), electroencephalography (EEG) and electroneuromyography (ENMG). Results: 245 patients were included in the analysis. One-hundred fourteen patients (47%) were admitted to the intensive care unit (ICU) and 10 (4%) died. The most frequently reported neuropsychiatric symptoms were motor deficit (41%), cognitive disturbance (35%), impaired consciousness (26%), psychiatric disturbance (24%), headache (20%) and behavioral disturbance (18%). The most frequent syndromes diagnosed were encephalopathy (43%), critical illness polyneuropathy and myopathy (26%), isolated psychiatric disturbance (18%), and cerebrovascular disorders (16%). No patients showed evidence of SARS-CoV-2 in their CSF. Encephalopathy was associated with greater age and higher risk of death. Critical illness neuromyopathy was associated with an extended stay in the ICU. Conclusions: The majority of the neuropsychiatric complications recorded could be imputed to critical illness, intensive care and systemic inflammation, which contrasts with the paucity of more direct SARS-CoV-2-related complications or post-infection disorders.

Direct visualization of native infectious SARS-CoV-2 ant its inactivation forms using high resolution Atomic Force Microscopy (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for COVID19, a new emerging pandemic affecting humans. Here, single viruses were analyze by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess infectious virus morphology in its native conformation, or upon inactivation treatments, at the nanoscale level and in 3D. High resolution AFM reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory, as revealed by combining AFM and plaque assays, to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-atomic force microscopy is a remarkable toolbox for rapid and direct virus identification and characterization.

Transferrin receptor is another receptor for SARS-CoV-2 entry (preprint)

Angiotensin-converting enzyme 2 (ACE2) has been suggested as a receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry to cause coronavirus disease 2019 (COVID-19). However, no ACE2 inhibitors have shown definite beneficiaries for COVID-19 patients, applying the presence of another receptor for SARS-CoV-2 entry. Here we show that ACE2 knockout dose not completely block virus entry, while TfR directly interacts with virus Spike protein to mediate virus entry and SARS-CoV-2 can infect mice with over-expressed humanized transferrin receptor (TfR) and without humanized ACE2. TfR-virus co-localization is found both on the membranes and in the cytoplasma, suggesting SARS-CoV-2 transporting by TfR, the iron-transporting receptor shuttling between cell membranes and cytoplasma. Interfering TfR-Spike interaction blocks virus entry to exert significant anti-viral effects. Anti-TfR antibody (EC50 16.6 nM) shows promising anti-viral effects in mouse model. Collectively, this report indicates that TfR is another receptor for SARS-CoV-2 entry and a promising anti-COVID-19 target.

SARS-CoV-2 Nucleocapsid protein attenuates stress granule formation and alters gene expression via direct interaction with host mRNAs (preprint)

The COVID-19 pandemic has caused over one million deaths thus far. There is an urgent need for the development of specific viral therapeutics and a vaccine. SARS-CoV-2 nucleocapsid (N) protein is highly expressed upon infection and is essential for viral replication, making it a promising target for both antiviral drug and vaccine development. Here, starting from a functional proteomics workflow, we initially catalogued the protein-protein interactions of 21 SARS-CoV-2 proteins in HEK293 cells, finding that the stress granule resident proteins G3BP1 and G3BP2 co-purify with N with high specificity. We demonstrate that N protein expression of in human cells sequesters G3BP1 and G3BP2 through its physical interaction with these proteins, attenuating stress granule (SG) formation. The ectopic expression of G3BP1 in N-expressing cells was sufficient to reverse this phenotype. Since N is an RNA-binding protein, we performed iCLIP- sequencing experiments in cells, with or without exposure to oxidative stress, to identify the host RNAs targeted by N. Our results indicate that SARS-CoV-2 N protein binds directly to thousands of host mRNAs under both conditions. Like the G3BPs stress granule proteins, N was found to predominantly bind its target mRNAs in their 3UTRs. RNA sequencing experiments indicated that expression of N results in wide-spread gene expression changes in both unstressed and oxidatively stressed cells. We suggest that N regulates host gene expression by both attenuating stress granules and binding directly to target mRNAs.

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Modified Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine (preprint)

The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Several vaccine candidates are currently being tested in the clinic. Inactivated virus and recombinant protein vaccines can be safe options but may require adjuvants to induce robust immune responses efficiently. In this work we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile). This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin. IMDQ-PEG-CHOL is used to induce a protective immune response against SARS-CoV-2 after single vaccination with trimeric recombinant SARS-CoV-2 spike protein in the BALB/c mouse model. Inclusion of amphiphilic IMDQ-PEG-CHOL in the SARS-CoV-2 spike vaccine formulation resulted in enhanced immune cell recruitment and activation in the draining lymph node. IMDQ-PEG-CHOL has a better safety profile compared to native soluble IMDQ as the former induces a more localized immune response upon local injection, preventing systemic inflammation. Moreover, IMDQ-PEG-CHOL adjuvanted vaccine induced enhanced ELISA and in vitro microneutralization titers, and a more balanced IgG2a/IgG1 response. To correlate vaccine responses with control of virus replication in vivo, vaccinated mice were challenged with SARS-CoV-2 virus after being sensitized by intranasal adenovirus-mediated expression of the human angiotensin converting enzyme 2 (ACE2) gene. Animals vaccinated with trimeric recombinant spike protein vaccine without adjuvant had lung virus titers comparable to non-vaccinated control mice, whereas animals vaccinated with IMDQ-PEG-CHOL-adjuvanted vaccine controlled viral replication and infectious viruses could not be recovered from their lungs at day 4 post infection. In order to test whether IMDQ-PEG-CHOL could also be used to adjuvant vaccines currently licensed for use in humans, proof of concept was also provided by using the same IMDQ-PEG-CHOL to adjuvant human quadrivalent inactivated influenza virus split vaccine, which resulted in enhanced hemagglutination inhibition titers and a more balanced IgG2a/IgG1 antibody response. Enhanced influenza vaccine responses correlated with better virus control when mice were given a lethal influenza virus challenge. Our results underscore the potential use of IMDQ-PEG-CHOL as an adjuvant to achieve protection after single immunization with recombinant protein and inactivated virus vaccines against respiratory viruses, such as SARS-CoV-2 and influenza viruses.

Potential Achilles heels of SARS-CoV-2 displayed by the base order-dependent component of RNA folding energy (preprint)

The energetics of the folding of a single-stranded nucleic acid into a stem-loop structure depend on both the composition and order of its bases. Composition tends to reflect genome-wide evolutionary pressures. Order better reflects local pressures. Base order is likely to be conserved when encoding a function critical for survival. The base order-dependent component of the folding energy has shown that a highly conserved region in HIV-1 genomes associates with an RNA structure. This corresponds to a packaging signal that is specifically recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a recently described antiviral compound (NSC 260594) or by synthetic oligonucleotides. Thus, a conserved base-order-rich region of HIV-1 may facilitate therapeutic attack. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e.g. NSP3, ORF3a), we suggest that their nucleic acid level functions, such as the ribosomal frameshifting element (FSE) that facilitates differential expression of 1a and 1ab polyproteins, can be considered potential "Achilles heels" for SARS-CoV-2 that should be susceptible to therapies like those envisaged for AIDS. The region of the FSE scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.