A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer–dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.

Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2

The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

Independent infections of porcine deltacoronavirus among Haitian children

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3–5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human–animal contact.

A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19.

Coinfection and Interference Phenomena Are the Results of Multiple Thermodynamic Competitive Interactions

Biological, physical and chemical interaction between one (or more) microorganisms and a host organism, causing host cell damage, represents an infection. Infection of a plant, animal or microorganism with a virus can prevent infection with another virus. This phenomenon is known as viral interference. Viral interference is shown to result from two types of interactions, one taking place at the cell surface and the other intracellularly. Various viruses use different receptors to enter the same host cell, but various strains of one virus use the same receptor. The rate of virus–receptor binding can vary between different viruses attacking the same host, allowing interference or coinfection. The outcome of the virus–virus–host competition is determined by the Gibbs energies of binding and growth of the competing viruses and host. The virus with a more negative Gibbs energy of binding to the host cell receptor will enter the host first, while the virus characterized by a more negative Gibbs energy of growth will overtake the host metabolic machine and dominate. Once in the host cell, the multiplication machinery is shared by the competing viruses. Their potential to utilize it depends on the Gibbs energy of growth. Thus, the virus with a more negative Gibbs energy of growth will dominate. Therefore, the outcome can be interference or coinfection, depending on both the attachment kinetics (susceptibility) and the intracellular multiplication machinery (permittivity). The ratios of the Gibbs energies of binding and growth of the competing viruses determine the outcome of the competition. Based on this, a predictive model of virus–virus competition is proposed.

The Impact of Angiotensin-Converting Enzyme 2 (ACE2) Expression Levels in Patients with Comorbidities on COVID-19 Severity: A Comprehensive Review

Angiotensin-Converting Enzyme 2 (ACE2) has been proved to be the main host cell receptor for the binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic. The SARS-CoV-2 spike (S) protein binds to ACE2 to initiate the process of replication. This enzyme is widely present in human organ tissues, such as the heart and lung. The pathophysiology of ACE2 in SARS-CoV-2 infection is complex and may be associated with several factors and conditions that are more severe in COVID-19 patients, such as age, male gender, and comorbidities, namely, cardiovascular diseases, chronic respiratory diseases, obesity, and diabetes. Here we present a comprehensive review that aims to correlate the levels of expression of the ACE2 in patients with comorbidities and with a poor outcome in COVID-19 disease. Significantly higher levels of expression of ACE2 were observed in myocardial and lung tissues in heart failure and COPD patients, respectively. An age-dependent increase in SARS2-CoV-2 receptors in the respiratory epithelium may be also responsible for the increased severity of COVID-19 lung disease in elderly people. Although the role of ACE2 is highlighted regarding the damage that can arise upon the SARS-CoV-2 invasion, there was no association observed between renin-angiotensin-aldosterone system (RAAS) inhibitors and the severity of COVID-19.

Mutations in two SARS-CoV-2 variants of concern reflect two distinct strategies of antibody escape

Understanding the factors that contribute to antibody escape of SARS-CoV-2 and its variants is key for the development of drugs and vaccines that provide broad protection against a variety of virus variants. Using microfluidic diffusional sizing, we determined the dissociation constant ((KD)) for the interaction between receptor binding domains (RBDs) of SARS-CoV-2 in its original version (WT) as well as alpha and beta variants with the host-cell receptor angiotensin converting enzyme 2 (ACE2). For RBD-alpha, the ACE2-binding affinity was increased by a factor of ten when compared with RBD-WT, while ACE2-binding of RBD-beta was largely unaffected. However, when challenged with a neutralizing antibody that binds to both RBD-WT and RBD-alpha with low nanomolar (KD) values, RBD-beta displayed no binding, suggesting a substantial epitope change. In SARS-CoV-2 convalescent sera, RBD-binding antibodies showed low nanomolar affinities to both wild-type and variant RBD proteins—strikingly, the concentration of antibodies binding to RBD-beta was half that of RBD-WT and RBD-alpha, again indicating considerable epitope changes in the beta variant. Our data therefore suggests that one factor contributing to the higher transmissibility and antibody evasion of SARS-CoV-2 alpha and beta is a larger fraction of viruses that can form a complex with ACE2. However, the two variants employ different mechanisms to achieve this goal. While SARS-CoV-2 alpha RBD binds with greater affinity to ACE2 and is thus more difficult to displace from the receptor by neutralizing antibodies, RBD-beta is less accessible to antibodies due to epitope changes which increases the chances of ACE2-binding and infection.

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies

Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.