Prototype mRNA vaccines imprint broadly neutralizing human serum antibodies after Omicron variant-matched boosting (preprint)

Immune imprinting is a phenomenon in which an individuals prior antigenic experiences influence responses to subsequent infection or vaccination. Here, using antibody depletion and multiplexed spike-binding assays, we characterized the type-specificity and cross-reactivity of serum antibody responses after mRNA vaccination in mice and human clinical trial participants. In mice, a single priming dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike minimally imprinted serum responses elicited by Omicron boosters, enabling a robust generation of type-specific antibodies. However, substantial imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron mRNA vaccine. In humans who received two BA.5 or XBB.1.5 Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with circulating Omicron variants as well as more distantly related sarbecoviruses. Because the serum neutralizing response against Omicron strains and other sarbecoviruses was completely abrogated after pre-clearing with the Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes shaped and shared by the antecedent mRNA-1273 primary series. Our depletion analysis also identified cross-reactive neutralizing antibodies that recognize distinct epitopes in the receptor binding domain (RBD) and S2 proteins with differential inhibitory effects on members of the sarbecovirus subgenus. Thus, although the serum antibody response to Omicron-based boosters in humans is dominantly imprinted by prior immunizations with prototype mRNA-1273 vaccines, this outcome can be beneficial as it drives expansion of multiple classes of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and extend activity to distantly related sarbecoviruses.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans (preprint)

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses of these vaccines and the development of new variant-derived ones. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells (MBCs). It remains unclear, however, whether the additional doses induce germinal centre (GC) reactions where reengaged B cells can further mature and whether variant-derived vaccines can elicit responses to novel epitopes specific to such variants. Here, we show that boosting with the original SARS-CoV-2 spike vaccine (mRNA-1273) or a B.1.351/B.1.617.2 (Beta/Delta) bivalent vaccine (mRNA-1273.213) induces robust spike-specific GC B cell responses in humans. The GC response persisted for at least eight weeks, leading to significantly more mutated antigen-specific MBC and bone marrow plasma cell compartments. Interrogation of MBC-derived spike-binding monoclonal antibodies (mAbs) isolated from individuals boosted with either mRNA-1273, mRNA-1273.213, or a monovalent Omicron BA.1-based vaccine (mRNA-1273.529) revealed a striking imprinting effect by the primary vaccination series, with all mAbs (n=769) recognizing the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted approach, we isolated mAbs that recognized the spike protein of the SARS-CoV-2 Omicron (BA.1) but not the original SARS-CoV-2 spike from the mRNA-1273.529 boosted individuals. The latter mAbs were less mutated and recognized novel epitopes within the spike protein, suggesting a naive B cell origin. Thus, SARS-CoV-2 boosting in humans induce robust GC B cell responses, and immunization with an antigenically distant spike can overcome the antigenic imprinting by the primary vaccination series.

Tracking infectious entry routes of SARS-CoV-2 (preprint)

SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity.

Multivalent designed proteins protect against SARS-CoV-2 variants of concern (preprint)

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness.

FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant (preprint)

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human natural defense mechanisms against SARS-CoV-2 are largely unknown. Serine proteases (SPs) including furin and TMPRSS2 cleave SARS-CoV-2 spike protein, facilitating viral entry. Here, we show that FXa, a SP for blood coagulation, is upregulated in COVID 19 patients compared to non-COVID-19 donors and exerts anti-viral activity. Mechanistically, FXa cleaves the SARS-CoV-2 spike protein, which prevents its binding to ACE2, and thus blocks viral entry. Furthermore, the variant B.1.1.7 with several mutations is dramatically resistant to the anti-viral effect of FXa compared to wild-type SARA-CoV-2 in vivo and in vitro. The anti-coagulant rivaroxaban directly inhibits FXa and facilitates viral entry, whereas the indirect inhibitor fondaparinux does not. In a lethal humanized hACE2 mouse model of SARS-CoV-2, FXa prolonged survival while combination with rivaroxaban but not fondaparinux abrogated this protection. These preclinical results identify a previously unknown SP function and associated anti-viral host defense mechanism and suggest caution in considering direct inhibitors for prevention or treatment of thrombotic complications in COVID-19 patients.

SARS-CoV-2 Spreads through Cell-to-Cell Transmission (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While ACE2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the variants of concern (VOC) B.1.1.7 and B.1.351 have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccine sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

A potently neutralizing anti-SARS-CoV-2 antibody inhibits variants of concern by binding a highly conserved epitope (preprint)

With the emergence of SARS-CoV-2 variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here we developed a panel of neutralizing anti-SARS-CoV-2 mAbs that bind the receptor binding domain of the spike protein at distinct epitopes and block virus attachment to cells and its receptor, human angiotensin converting enzyme-2 (hACE2). While several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by historical SARS-CoV-2 strains, others induced escape variants in vivo and lost activity against emerging strains. We identified one mAb, SARS2-38, that potently neutralizes all SARS-CoV-2 variants of concern tested and protects mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engages a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of inhibitory antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody (preprint)

The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2X259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2X259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2X259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.

A public vaccine-induced human antibody protects against SARS-CoV-2 and emerging variants (preprint)

The emergence of antigenically distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility is a public health threat. Some of these variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies, which principally target the receptor binding domain (RBD) on the virus spike glycoprotein. Here, we describe 2C08, a SARS-CoV-2 mRNA vaccine-induced germinal center B cell-derived human monoclonal antibody that binds to the receptor binding motif within the RBD. 2C08 broadly neutralizes SARS-CoV-2 variants with remarkable potency and reduces lung inflammation, viral load, and morbidity in hamsters challenged with either an ancestral SARS-CoV-2 strain or a recent variant of concern. Clonal analysis identified 2C08-like public clonotypes among B cell clones responding to SARS-CoV-2 infection or vaccination in at least 20 out of 78 individuals. Thus, 2C08-like antibodies can be readily induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

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.

Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants. (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.

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.

in vitro: Natural Compounds (Thymol, Carvacrol, Hesperidine, And Thymoquinone) Against SARS-CoV2 Strain Isolated From Egyptian Patients (preprint)

The current pandemic of the coronavirus disease-2019 (COVID-19) has badly affected our life during the year 2020. SARS-CoV-2 is the primary causative agent of the newly emerged pandemic. Natural flavonoids, Terpenoid and Thymoquinone are tested against different viral and host-cell protein targets. These natural compounds have a good history in treating Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Molecular docking combined with cytotoxicity and plaque reduction assay is used to test the natural compounds against different viral (Spike, RdRp, and Mpro) and host-cell (TMPRSS II, keap 1, and ACE2) targets. The results demonstrate the binding possibility of the natural compounds (Thymol, Carvacrol, Hesperidine, and Thymoquinone) to the viral main protease (Mpro). Some of these natural compounds were approved to start clinical trail from Egypt Center for Research and Regenerative Medicine ECRRM IRB (Certificate No.IRB00012517)

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.

Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects naive mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

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.