TCR transgenic clone selection guided by immune receptor analysis and single cell RNA expression of polyclonal responders (preprint)

Since the precursor frequency of naive T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are PCR-cloned into expression vectors. Here, we exploited the rapid advances in single cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice (CORona Spike Epitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation and immunophenotype in vivo. In contrast, a traditional method based on hybridoma technology was unsuccessful. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.

A Complement Atlas identifies interleukin 6 dependent alternative pathway dysregulation as a key druggable feature of COVID-19 (preprint)

To improve COVID-19 therapy, it is essential to understand the mechanisms driving critical illness. The complement system is an essential part of innate host defense that can also contribute to injury. All complement pathways have been implicated in COVID-19 pathogenesis, however the upstream drivers and downstream consequences on tissue injury remain ill-defined. Here, we demonstrate that complement activation is mediated by the alternative pathway and we provide a comprehensive atlas of the alterations in complement around the time of respiratory deterioration. Proteome and single-cell sequencing mapping across cell types and tissues reveals a division of labor between lung epithelial, stromal and myeloid cells in the production of complement, in addition to liver-derived factors. Upstream, IL-6 drives complement responses, linking complement dysregulation to approved COVID-19 therapies. In an exploratory proteomic study, C5 inhibition improves epithelial damage and markers of disease severity. Collectively, these results identify complement dysregulation as a key druggable feature of COVID-19.

Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that bind a conserved membrane proximal epitope of the spike (preprint)

Currently circulating SARS-CoV-2 variants have gained complete or significant resistance to all SARS-CoV-2-neutralizing antibodies that have been used in the clinic. Such antibodies can prevent severe disease in SARS-CoV-2 exposed patients for whom vaccines may not provide optimal protection. Here, we describe single-domain antibodies (VHHs), also known as nanobodies, that can broadly neutralize SARS-CoV-2 with unusually high potency. Structural analysis revealed their binding to a unique, highly conserved, membrane proximal, quaternary epitope in the S2 subunit of the spike. Furthermore, a VHH-human IgG1 Fc fusion, efficiently expressed in Chinese hamster ovary cells as a stable antibody construct, protected hamsters against SARS-CoV-2 replication in a therapeutic setting when administered systemically at low dose. This VHH-based antibody represents a new candidate anti-COVID-19 biologic that targets the Achilles heel of the viral spike.

Efficacy and safety of the investigational complement C5 inhibitor zilucoplan in patients hospitalized with Covid-19: an open-label randomized controlled trial (preprint)

Background: The efficacy and safety of complement inhibition in Covid-19 patients is unclear.Methods: A multicenter randomized controlled, open-label trial. Hospitalized Covid-19 patients with signs of hyperinflammation and hypoxemia (PaO2/FiO2 below 350 mHg) were randomized (2:1 ratio) to receive standard of care with or without the C5 inhibitor zilucoplan daily for 14 days, under antibiotic prophylaxis. The primary outcome was improvement in oxygenation at day 6 and 15.Results: 81 patients were randomly assigned to zilucoplan (n=55) or the control group (n=26). 78 patients were included in the safety and primary analysis. Most  were men (87%) and the median age was 63 years. The mean improvement in PaO2/FiO2 from baseline to day 6 was 56·4 mmHg in the zilucoplan group and 20·6 mmHg in the control group (mean difference +35.8; 95% confidence interval (CI) -9.4 to 80.9; p=0.12), an effect also observed  at day 15. Day 28 mortality was 9% in the zilucoplan and 21% in the control group (odds ratio 0·4; 95% CI 0·1 to 1·5) At long-term follow up, the distance walked in a 6 min test was 539·7 m in zilucoplan and 490·6 m in the control group (p=0·18). Zilucoplan lowered serum C5b-9 (p<0·001) and interleukin-8 (p=0·03) concentration compared with control. No relevant safety differences between the zilucoplan and control group were identified.Conclusion: Administration of zilucoplan to Covid-19 patients in this proof-of-concept randomized trial was well tolerated under antibiotic prophylaxis. While not reaching statistical significance, indicators of respiratory function and clinical outcome suggest that C5 inhibition might be beneficial, although this requires further research in larger randomized studies.(Funded by UCB and Ghent University Special Research Fund for COVID-19 Research; ClinicalTrials.gov, NCT04382755 – May 11, 2020; EudraCT, 2020-002130-33 – May 6, 2020)

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.