Temporal Increase in Neutralization Potency of SARS-CoV-2 Antibodies and Reduced Viral Variant Escape after Sputnik V Vaccination (preprint)

Recent studies have shown a temporal increase in the neutralizing antibody potency and breadth to SARS-CoV-2 variants in coronavirus disease 2019 (COVID-19) convalescent individuals. Here, we observed a similar process after Sputnik V vaccination. We examined the longitudinal antibody responses and viral neutralizing capacity to variants of concern (VOCs: Alpha, Beta, Gamma, and Delta) and a broadly spread variant of interest (VOI: Lambda) in volunteers up to 6 months after receiving the Sputnik V vaccine in Argentina. A collection of 1,800 serum samples obtained between January and August 2021 was used. The analysis indicates that while anti-spike IgG levels significantly wane over time, the neutralizing potency to the first-wave linages of SARS-CoV-2 and VOC increases within four months of vaccination, suggesting that antibody maturation occurs. This increase was more evident for the Beta and Gamma variants, which showed the highest propensity for neutralization escape. Our observations suggest that protection increases over the six months following vaccination as a consequence of antibody maturation, resulting in improved potency of antibodies to viral escape mutations.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell (preprint)

Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2 (preprint)

SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization (preprint)

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of most COVID-19 vaccines and being developed as therapeutics, escape mutations could compromise such countermeasures. To define the immune-mediated mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor binding domain (RBD) to generate 48 escape mutants. These variants were mapped onto the RBD structure and evaluated for cross-resistance by convalescent human plasma. Although each mAb had unique resistance profiles, many shared residues within an epitope, as several variants were resistant to multiple mAbs. Remarkably, we identified mutants that escaped neutralization by convalescent human sera, suggesting that some humans induce a narrow repertoire of neutralizing antibodies. By comparing the antibody-mediated mutational landscape in S protein with sequence variation in circulating SARS-CoV-2 strains, we identified single amino acid substitutions that could attenuate neutralizing immune responses in some humans.

Reanalysis of MERS, SARS and COVID-19 Infection Datasets using VirOmics Playground Reveals Common Patterns in Gene and Protein Expression (preprint)

Three lethal lower respiratory tract coronavirus epidemics have occurred over the past 20 years. This coincided with major developments in genome-wide gene and protein expression analysis, resulting in a wealth of datasets in the public domain. Seven such in vitro studies were selected for comparative bioinformatic analysis through the VirOmics Playground, a user-friendly visualisation and exploration platform we recently developed. Despite the heterogeneous nature of the data sets, several commonalities could be observed across studies and species. Differences, on the other hand, reflected not only variations between species, but also other experimental variables, such as cell lines used for the experiments, infection protocols and potential discrepancies between transcriptome and proteome data. The results presented here are available online and can be replicated through the VirOmics Playground.

SARS-CoV-2 and Malayan pangolin coronavirus infect human endoderm, ectoderm and induced lung progenitor cells (preprint)

Since the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in several somatic cells, little is known about the infection of SASRS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that SARS-CoV-2 pseudovirus and GX_P2V could infect lung progenitor and even anterior foregut endoderm cells causing these cells death, which differentiated from human embryonic stem cells (hESCs). The infection and replication of SARS-CoV-2 and GX_P2V were inhibited when treated with whey protein of breastmilk and Remdesivir, confirming that these two viruses could infect lung progenitor and even anterior foregut endoderm. Moreover, we found that SARS-CoV-2 pseudovirus could infect endoderm and ectoderm. We found that whey protein blocked SARS-CoV-2 infecting these cells. In line with the SARS-CoV-2 results, GX_P2V could also infected endoderm and ectoderm, and also was inhibited by Remdesivir treatment. Although expressing coronavirus related receptor such as ACE2 and TMPRSS2, mesoderm cells are not permissive for SARS-CoV-2 and GX_P2V infection, which needed further to study the mechanisms. Interestingly, we also found that hESCs, which also express ACE2 and TMPRSS2 markers, are permissive for GX_P2V but not SARS-CoV-2 pseudovirus infection and replication, indicating the widespread cell types for GX_P2V infection. Heparin treatment blocked efficiently viral infection. These results provided insight that these stem cells maybe provided a stable repository of coronavirus function or genome. The potential consequence of SARS-CoV-2 and animal coronavirus such as GX_P2V infection in hESCs, germ layer and induced progenitors should be closely monitored.

Nitazoxanide and JIB-04 have broad-spectrum antiviral activity and inhibit SARS-CoV-2 replication in cell culture and coronavirus pathogenesis in a pig model (preprint)

Pathogenic coronaviruses represent a major threat to global public health. Here, using a recombinant reporter virus-based compound screening approach, we identified several small-molecule inhibitors that potently block the replication of the newly emerged severe acute respiratory syndrome virus 2 (SARS-CoV-2). Two compounds, nitazoxanide and JIB-04 inhibited SARS-CoV-2 replication in Vero E6 cells with an EC50 of 4.90 M and 0.69 M, respectively, with specificity indices of greater than 150. Both inhibitors had in vitro antiviral activity in multiple cell types against some DNA and RNA viruses, including porcine transmissible gastroenteritis virus. In an in vivo porcine model of coronavirus infection, administration of JIB-04 reduced virus infection and associated tissue pathology, which resulted in improved body weight gain and survival. These results highlight the potential utility of nitazoxanide and JIB-04 as antiviral agents against SARS-CoV-2 and other viral pathogens.

Molecular basis for SARS-CoV-2 spike affinity for human ACE2 receptor (preprint)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused substantially more infections, deaths, and economic disruptions than the 2002-2003 SARS-CoV. The key to understanding SARS-CoV-2's higher infectivity may lie in its host receptor recognition mechanism. This is because experiments show that the human ACE2 protein, which serves as the primary receptor for both CoVs, binds to CoV-2's spike protein 5-20 fold stronger than SARS-CoV's spike protein. The molecular basis for this difference in binding affinity, however, remains unexplained and, in fact, a comparison of X-ray structures leads to an opposite proposition. To gain insight, we use all-atom molecular dynamics simulations. Free energy calculations indicate that CoV-2's higher affinity is due primarily to differences in specific spike residues that are local to the spike-ACE2 interface, although there are allosteric effects in binding. Comparative analysis of equilibrium simulations reveals that while both CoV and CoV-2 spike-ACE2 complexes have similar interfacial topologies, CoV-2's spike protein engages in greater numbers, combinatorics and probabilities of hydrogen bonds and salt bridges with ACE2. We attribute CoV-2's higher affinity to these differences in polar contacts, and these findings also highlight the importance of thermal structural fluctuations in spike-ACE2 complexation. We anticipate that these findings will also inform the design of spike-ACE2 peptide blockers that, like in the cases of HIV and Influenza, can serve as antivirals.

Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition (preprint)

Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails--including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion (preprint)

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG) that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an ISG screen against VSV-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of virus replication. Mechanistically, internalized 25HC accumulates in the late endosomes and blocks cholesterol export, thereby restricting SARS-CoV-2 spike protein catalyzed membrane fusion. Our results highlight a unique antiviral mechanism of 25HC and provide the molecular basis for its possible therapeutic development.

Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2. (preprint)

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune {gamma}-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.

TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes (preprint)

Both gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA have been frequently observed in COVID-19 patients. However, whether SARS-CoV-2 replicate in the human intestine and its clinical relevance to potential fecal-oral transmission remain unclear. Here, we demonstrate productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. In addition to TMPRSS2, another mucosa-specific serine protease, TMPRSS4, also enhanced SARS-CoV-2 spike fusogenic activity and mediated viral entry into host cells. However, newly synthesized viruses released into the intestinal lumen were rapidly inactivated by human colonic fluids and no infectious virus was recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

Inhibition of PIKfyve kinase prevents infection by EBOV and SARS-CoV-2 (preprint)

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric VSV containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or SARS-CoV-2 (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small molecule inhibitors of the main endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define new tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.