SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive ...possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.
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.
Secretion of outer membrane vesicles (OMV) is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella ...catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig) D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR) clustering and Ca(2+) mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+) IgD(+) lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86), whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.
The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence ...responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.
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•IGH genes with high allelic diversity are used in anti-SARS-CoV-2 antibodies•IGH allele usage can influence the activity of neutralizing antibodies•Cryo-EM analysis confirms the role of germline-encoded residues in antigen binding•IGH genotyping can uncover differences in Ab responses due to allelic variation
The genes and alleles of the antigen receptor loci are highly variable between individuals, which may affect the quality of the immune response to different pathogens. Here, Pushparaj et al. use immunoglobulin genotyping and monoclonal antibody engineering to illustrate how heritable differences in such genes can modulate anti-SARS-CoV-2 antibody function.
Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased ...valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC
in the low picomolar range.
The outbreak and spread of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) is a current global health emergency, and effective prophylactic vaccines are needed urgently. The spike ...glycoprotein of SARS-CoV-2 mediates entry into host cells, and thus is the target of neutralizing antibodies. Here, we show that adjuvanted protein immunization with soluble SARS-CoV-2 spike trimers, stabilized in prefusion conformation, results in potent antibody responses in mice and rhesus macaques, with neutralizing antibody titers exceeding those typically measured in SARS-CoV-2 seropositive humans by more than one order of magnitude. Neutralizing antibody responses were observed after a single dose, with exceptionally high titers achieved after boosting. A follow-up to monitor the waning of the neutralizing antibody responses in rhesus macaques demonstrated durable responses that were maintained at high and stable levels at least 4 months after boosting. These data support the development of adjuvanted SARS-CoV-2 prefusion-stabilized spike protein subunit vaccines.
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Neutralizing antibodies are detectable after one adjuvanted spike protein immunizationBoosting yields higher neutralizing titers than observed in seropositive human donorsNeutralizing antibody titers remain high for at least 4 months after the last boostImmunization stimulates spike-specific memory B cells
Mandolesi et al. show that immunization with adjuvanted prefusion-stabilized SARS-CoV-2 spike glycoprotein yields potent antibody responses in mice and macaques. Neutralizing antibodies are detectable after one immunization. Boosting results in exceptional potency, with neutralizing titers exceeding (>10-fold) those observed in seropositive humans and remaining high during a 4-month follow-up.
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with resistance to neutralizing antibodies are threatening to undermine vaccine efficacy. Vaccination and ...infection have led to widespread humoral immunity against the pandemic founder (Wu-Hu-1). Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet clear whether heterotypic boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. We show, in macaques immunized with Wu-Hu-1 spike, that a single dose of adjuvanted beta variant receptor binding domain (RBD) protein broadens neutralizing antibody responses to heterologous VOCs. Passive transfer of plasma sampled after Wu-Hu-1 spike immunization only partially protects K18-hACE2 mice from lethal challenge with a beta variant isolate, whereas plasma sampled following heterotypic RBD boost protects completely against disease.
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•Heterotypic RBD boost elicits cross-neutralizing antibody responses in macaques•No evidence that original antigenic sin hinders booster immunizations with beta RBD•Pre-boost plasma only partially protects K18-hACE2 mice from beta variant challenge•Post-boost plasma affords full protection from beta variant challenge
The emergence and spread of antibody-resistant SARS-CoV-2 variants of concern (VOCs) threatens to diminish vaccine efficacy. Sheward et al. show, in rhesus macaques and K18-hACE2 mice, that reduced vaccine protection against VOCs can be restored by broadening antibody responses with a third, heterotypic RBD booster immunization.
Moraxella catarrhalis is an emerging human-specific pathogen responsible for upper and lower respiratory tract infections. Understanding the events in the complex pathogenesis and underlying ...mechanisms during M. catarrhalis infection is a key to the development of novel therapeutics and vaccines.
Several novel findings have been reported on Moraxella pathogenesis and, in parts, explain how the species stands as a commensal in preschool children and survives in the host. Molecular structures for different adhesins in addition to target ligands with respect to signalling and invasion have been defined. Evasion of the complement system allows Moraxella to survive in the mucosa and by neutralizing alpha1-antichymotrypsin the protease activity is increased, resulting in tissue destruction and thus promotion of bacterial attachment. Moraxella-dependent cell activation via immunoglobulin D in addition to toll-like receptors and specific epithelial cell inhibition by cross-linking of carcinoembryonic antigen-related cell adhesion molecule-1 in the early innate immune response and, finally, the ability of M. catarrhalis to form biofilms are other specific research areas of interest.
Recent advances have allowed a more detailed picture of the processes involved in bacteria-host cell interactions, the cause of inflammatory processes and specific host defense responses against the intriguing species Moraxella.
Autophagy, a highly regulated degradative process that promotes cellular homeostasis, is increasingly recognised as a fundamental component of the cellular response against viral infection. In this ...study, we investigated the role of autophagy during Junín virus (JUNV) multiplication using human A549 cells. We found that JUNV infection induces an increment of the LC3-II/LC3-I ratio, an accumulation of punctate pattern in RFP-LC3-transfected cells and the colocalisation of viral nucleoprotein and LC3 protein, suggesting autophagosome formation. JUNV infection also induced the degradation of the autophagy receptor p62, suggesting that complete autophagic flux was triggered. In addition, we showed that inhibition of autophagy with bafilomycin A1 or 3-methyladenine significantly reduces viral multiplication. Moreover, viral yield was increased when autophagy was induced using rapamycin. Furthermore, JUNV infection induced the colocalisation of p62, ATG16, RAB5, RAB7A and LAMP1 with the autophagosomal LC3 protein. That suggests that phagosomes undergo the maturation process during viral infection. Finally, we demonstrated that siRNA experiments targeting essential autophagy genes (ATG5, ATG7 and Beclin 1) reduce viral protein synthesis and viral yield. Overall, our results indicate that JUNV activates host autophagy machinery enhancing its multiplication.
Moraxella catarrhalis is an emerging human respiratory pathogen in patients with chronic obstructive pulmonary disease (COPD) and in children with acute otitis media. The specific secretion machinery ...known as outer membrane vesicles (OMVs) is a mechanism by which Gram-negative pathogens interact with host cells during infection. We identified 57 proteins in M. catarrhalis OMVs using a proteomics approach combining two-dimensional SDS-PAGE and MALDI-TOF mass spectrometry analysis. The OMVs contained known surface proteins such as ubiquitous surface proteins (Usp) A1/A2, and Moraxella IgD-binding protein (MID). Most of the proteins are adhesins/virulence factors triggering the immune response, but also aid bacteria to evade the host defence. FITC-stained OMVs bound to lipid raft domains in alveolar epithelial cells and were internalized after interaction with Toll-like receptor 2 (TLR2), suggesting a delivery to the host tissue of a large and complex group of OMV-attributed proteins. Interestingly, OMVs modulated the pro-inflammatory response in epithelial cells, and UspA1-bearing OMVs were found to specifically downregulate the reaction. When mice were exposed to OMVs, a pulmonary inflammation was clearly seen. Our findings indicate that Moraxella OMVs are highly biologically active, transport main bacterial virulence factors and may modulate the epithelial pro-inflammatory response.
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