Identification of new drugs to counteract anti-spike IgG-induced hyperinflammation in severe COVID-19 (preprint)

Previously, we and others have shown that SARS-CoV-2 spike-specific IgG antibodies play a major role in disease severity in COVID-19 by triggering macrophage hyperactivation, disrupting endothelial barrier integrity, and inducing thrombus formation. This hyperinflammation is dependent on high levels of anti-spike IgG with aberrant Fc tail glycosylation, leading to Fc{gamma} receptor hyperactivation. For development of immune-regulatory therapeutics, drug specificity is crucial to counteract excessive inflammation while simultaneously minimizing inhibition of antiviral immunity. We here developed an in vitro activation assay to screen for small molecule drugs that specifically counteract antibody-induced pathology. We identified that anti-spike induced inflammation is specifically blocked by small molecule inhibitors against SYK and PI3K. We identified SYK inhibitor entospletinib as the most promising candidate drug, which also counteracted anti-spike-induced endothelial dysfunction and thrombus formation. Moreover, entospletinib blocked inflammation by different SARS-CoV-2 variants of concern. Combined, these data identify entospletinib as a promising treatment for severe COVID-19.

Broad SARS-CoV-2 Neutralization by Monoclonal and Bispecific Antibodies Derived from a Gamma-infected Individual (preprint)

The worldwide pandemic caused by SARS-CoV-2 has remained a human medical threat due to the continued evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants for therapeutic and prophylactic use. A stabilized autologous SARS-CoV-2 spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants of concern, with COVA309-35 being the most potent against the autologous virus, as well as against Omicron BA.1 and BA.2. When combining the COVA309 mAbs as cocktails or bispecific antibody formats, the breadth and potency was significantly improved against all tested variants. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

The SARS-CoV-2 spike N-terminal domain engages 9-O-acetylated α2-8-linked sialic acids (preprint)

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD) which, in other coronaviruses, includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of functionality. To analyze gain or loss of glycan-binding in VoC, trimeric fluorescent NTD proteins were used. Binding properties were analyzed biochemically on Vero E6 cells and tissue samples. Unexpectedly, the SARS-CoV-2 Beta (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. This demonstrates plasticity for improved engagement to sialic acids, possibly under immunological pressure.

IgG1 responses following SARS-CoV-2 infection are polyclonal and highly personalized, whereby each donor and each clone displays a distinct pattern of cross-reactivity against SARS-CoV-2 variants (preprint)

Using a recently introduced efficient mass spectrometry-based approach we monitored in molecular detail the IgG1 clonal responses in individual donors' IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.

Mapping the antigenic diversification of SARS-CoV-2 (preprint)

Large-scale vaccination campaigns have prevented countless SARS-CoV-2 infections, hospitalizations and deaths. However, the emergence of variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similar to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants-of-concern (VOCs) of a unique set of sera from patients infected with a range of VOCs. Infections with ancestral or Alpha strains induced the broadest immunity, while individuals infected with other VOCs had more strain-specific responses. Omicron was substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that all VOCs preceding Omicron belong to one antigenic cluster, while Omicron forms a new antigenic cluster associated with immune escape and likely requiring vaccine updates to ensure vaccine effectiveness.

A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike (preprint)

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that ~82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells (~0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched (~0.05%) and unswitched B cells (~0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

Four SARS-CoV-2 vaccines induce quantitatively different antibody responses against SARS-CoV-2 variants (preprint)

BackgroundEmerging and future SARS-CoV-2 variants may jeopardize the effectiveness of vaccination campaigns. Therefore, it is important to know how the different vaccines perform against diverse SARS-CoV-2 variants. MethodsIn a prospective cohort of 165 SARS-CoV-2 naive health care workers, vaccinated with either one of four vaccines (BNT162b2, mRNA-1273, AZD1222 or Ad26.COV2.S), we performed a head-to-head comparison of the ability of sera to recognize and neutralize SARS-CoV-2 variants of concern (VOCs; Alpha, Beta, Gamma, Delta and Omicron). Repeated serum sampling was performed 5 times during a year (from January 2021 till January 2022), including before and after booster vaccination with BNT162b2. FindingsFour weeks after completing the initial vaccination series, SARS-CoV-2 wild-type neutralizing antibody titers were highest in recipients of BNT162b2 and mRNA-1273 (geometric mean titers (GMT) of 197 [95% CI 149-260] and 313 [95% CI 218-448], respectively), and substantially lower in those vaccinated with the adenovirus vector-based vaccines AZD1222 and Ad26.COV2.S (GMT of 26 [95% CI 18-37] and 14 [95% CI 8-25] IU/ml, respectively). These findings were robust for adjustment to age and sex. VOCs neutralization was reduced in all vaccine groups, with the largest (9- to 80-fold) reduction in neutralization observed against the Omicron variant. The booster BNT162b2 vaccination increased neutralizing antibody titers for all groups with substantial improvement against the VOCs including the Omicron variant. Study limitations include the lack of cellular immunity data. ConclusionsOverall, this study shows that the mRNA vaccines appear superior to adenovirus vector-based vaccines in inducing neutralizing antibodies against VOCs four weeks after initial vaccination and after booster vaccination.

Probing Affinity, Avidity, Anti-Cooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses (preprint)

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behaviour of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behaviour arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e. Fabs). Surprisingly, these sub-stoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.

Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and vaccination (preprint)

Emerging SARS-CoV-2 variants pose a threat to human immunity induced by natural infection and vaccination. We assessed the recognition of three variants of concern (B.1.1.7, B.1.351 and P.1) in cohorts of COVID-19 patients ranging in disease severity (n = 69) and recipients of the Pfizer/BioNTech vaccine (n = 50). Spike binding and neutralization against all three VOC was substantially reduced in the majority of samples, with the largest 4-7-fold reduction in neutralization being observed against B.1.351. While hospitalized COVID-19 patients and vaccinees maintained sufficient neutralizing titers against all three VOC, 39% of non-hospitalized patients did not neutralize B.1.351. Moreover, monoclonal neutralizing antibodies (NAbs) show sharp reductions in their binding kinetics and neutralizing potential to B.1.351 and P.1, but not to B.1.1.7. These data have implications for the degree to which pre-existing immunity can protect against subsequent infection with VOC and informs policy makers of susceptibility to globally circulating SARS-CoV-2 VOC.

Single-dose SARS-CoV-2 vaccine in a prospective cohort of COVID-19 patients (preprint)

Background The urgent need for, but limited availability of, SARS-CoV-2 vaccines worldwide has led to widespread consideration of dose sparing strategies, particularly single vaccine dosing of individuals with prior SARS-CoV-2 infection. Methods We evaluated SARS-CoV-2 specific antibody responses following a single-dose of BNT162b2 (Pfizer-BioNTech) mRNA vaccine in 155 previously SARS-CoV-2-infected individuals participating in a population-based prospective cohort study of COVID-19 patients. Participants varied widely in age, comorbidities, COVID-19 severity and time since infection, ranging from 1 to 15 months. Serum antibody titers were determined at time of vaccination and one week after vaccination. Responses were compared to those in SARS-CoV-2-naive health care workers after two BNT162b2 mRNA vaccine doses. Results Within one week of vaccination, IgG antibody levels to virus spike and RBD proteins increased 27 to 29-fold and neutralizing antibody titers increased 12-fold, exceeding titers of fully vaccinated SARS-CoV-2-naive controls (95% credible interval (CrI): 0.56 to 0.67 v. control 95% CrI: -0.16 to -0.02). Pre-vaccination neutralizing antibody titers had the largest positive mean effect size on titers following vaccination (95% CrI (0.16 to 0.45)). COVID-19 severity, the presence of comorbidities and the time interval between infection and vaccination had no discernible impact on vaccine response. Conclusion A single dose of BNT162b2 mRNA vaccine up to 15 months after SARS-CoV-2 infection provides neutralizing titers exceeding two vaccine doses in previously uninfected individuals. These findings support wide implementation of a single-dose mRNA vaccine strategy after prior SARS-CoV-2 infection.

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)

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection (preprint)

The SARS-CoV-2 pandemic is continuing to disrupt personal lives, global healthcare systems and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits and cynomolgus macaques. The vaccine-induced immunity protected macaques against a high dose challenge, resulting in strongly reduced viral infection and replication in upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Cross-neutralization of a SARS-CoV-2 antibody to a functionally conserved site is mediated by avidity (preprint)

Most antibodies isolated from COVID-19 patients are specific to SARS-CoV-2. COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here we determined a crystal structure of COVA1-16 Fab with the SARS-CoV-2 RBD, and a negative-stain EM reconstruction with the spike glycoprotein trimer, to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long CDR H3, and competes with ACE2 binding due to steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with structural and functional rationale for the epitope conservation, provide a blueprint for development of more universal SARS-like coronavirus vaccines and therapies.

An alternative binding mode of IGHV3-53 antibodies to the SARS-CoV-2 receptor binding domain (preprint)

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization.