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.

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 Ca²⁺ 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. Outer membrane vesicles secreted by pathogenic bacteria are recognized as a long-distance delivery system which transports diverse virulence factors, and allows pathogens to interact with the host, and hence the possibility to modify the immune response without close contact. Our study shows that Moraxella catarrhalis outer membrane vesicles that also exist in patients can activate human B cells isolated from pharyngeal lymphoid tissue. These findings have significant implications for understanding Moraxella pathogenesis since palatine tonsils are a potential tissue reservoir. Vesicles secreted by Moraxella bind to tonsillar B cells through the superantigen Moraxella IgD-binding protein designated MID. The interaction between MID and the B cell receptor induces Ca²⁺ mobilization and receptor clustering in lipid raft motifs followed by internalization of vesicles. Mainly Toll-like receptor 9, a pathogen recognition receptor of the innate immune system, participates in the signaling induced by vesicles through sensing of DNA associated with vesicles. The vesicle-dependent B cell activation induces up-regulation of surface activation markers in addition to IL-6 and IgM secretion. Vesicle secretion provides Moraxella with a sophisticated mechanism to modify the host immune response, avoiding contact between bacteria and the host.

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.

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.

AlthoughBordetella pertussishas been observed to survive inside macrophages, its ability to resist or evade degradation in phagolysosomes has not been defined. We here investigated the trafficking ofB. pertussisupon entry into human macrophages. During the first hours following phagocytosis, a high percentage of bacteria were destroyed within acidic compartments positive for the lysosome-associated membrane proteins (LAMP). However, roughly one-fourth of the bacteria taken up evade this initial killing event, remaining in nonacidic compartments. Forty-eight hours after infection, the number of intracellular bacteria per cell increased, suggesting thatB. pertussisis capable of replicating in this type of compartment. Viable bacteria accumulated within phagosomal compartments positive for the early endosomal marker Rab5 but not the late endosomal marker LAMP. Moreover,B. pertussis-containing phagosomes acquired exogenously added transferrin, indicating that intracellular bacteria have access to extracellular components and essential nutrients via the host cell recycling pathway. Overall, these results suggest thatB. pertussissurvives and eventually replicates in compartments with characteristics of early endosomes, potentially contributing to its extraordinary ability to persist within hosts and populations.

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 α1-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.