Search

Conventional approaches to isolate and characterize nanobodies are laborious. We combine phage display, multivariate enrichment, next-generation sequencing, and a streamlined screening strategy to identify numerous anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nanobodies. We characterize their potency and specificity using neutralization assays and hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most potent nanobodies bind to the receptor binding motif of the receptor binding domain (RBD), and we identify two exceptionally potent members of this category (with monomeric half-maximal inhibitory concentrations 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.

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.

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., Graphical Abstract, 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.

The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet known whether heterotypic vaccine 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 primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein. Passive immunization with plasma sampled following this boost protected K18-hACE2 mice from lethal challenge with a 501Y.V2 clinical isolate, whereas only partial protection was afforded by plasma sampled after two Wu-Hu-1 spike immunizations.

The outbreak and spread of SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2), the cause of coronavirus disease 2019 (COVID-19), is a current global health emergency and a prophylactic vaccine is needed urgently. The spike glycoprotein of SARS-CoV-2 mediates entry into host cells, and thus is a target for neutralizing antibodies and vaccine design. Here we show that adjuvanted protein immunization with SARS-CoV-2 spike trimers, stabilized in prefusion conformation, results in potent antibody responses in mice and rhesus macaques with neutralizing antibody titers orders of magnitude greater than those typically measured in serum from SARS-CoV-2 seropositive humans. Neutralizing antibody responses were observed after a single dose, with exceptionally high titers achieved after boosting. Furthermore, neutralizing antibody titers elicited by a dose-sparing regimen in mice were similar to those obtained from a high dose regimen. Taken together, these data strongly support the development of adjuvanted SARS-CoV-2 prefusion-stabilized spike protein subunit vaccines.

Antibody responses vary widely between individuals1, complicating the correct classification of low-titer measurements using conventional assay cut-offs. We found all participants in a clinically diverse cohort of SARS-CoV-2 PCR+ individuals (n=105) – and n=33 PCR+ hospital staff – to have detectable IgG specific for pre-fusion-stabilized spike (S) glycoprotein trimers, while 98% of persons had IgG specific for the receptor-binding domain (RBD). However, anti-viral IgG levels differed by several orders of magnitude between individuals and were associated with disease severity, with critically ill patients displaying the highest anti-viral antibody titers and strongest in vitro neutralizing responses. Parallel analysis of random healthy blood donors and pregnant women (n=1,000) of unknown serostatus, further demonstrated highly variable IgG titers amongst seroconverters, although these were generally lower than in hospitalized patients and included several measurements that scored between the classical 3 and 6SD assay cut-offs. Since the correct classification of seropositivity is critical for individual- and population-level metrics, we compared different probabilistic algorithms for their ability to assign likelihood of past infection. To do this, we used tandem anti-S and -RBD IgG responses from our PCR+ individuals (n=138) and a large cohort of historical negative controls (n=595) as training data, and generated an equal-weighted learner from the output of support vector machines and linear discriminant analysis. Applied to test samples, this approach provided a more quantitative way to interpret anti-viral titers over a large continuum, scrutinizing measurements overlapping the negative control background more closely and offering a probability-based diagnosis with potential clinical utility. Especially as most SARS-CoV-2 infections result in asymptomatic or mild disease, these platform-independent approaches improve individual and epidemiological estimates of seropositivity, critical for effective management of the pandemic and monitoring the response to vaccination.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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 [alpha]1-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. SUMMARY: 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. (Less)

Expression of transcripts for the homotypic adhesion protein epithelial V-like antigen 1 (EVA1), also known as myelin protein zero like-2 (Mpzl2), is known to be present in thymic stromal cells. However, protein expression within different thymic subsets, stromal and/or lymphoid, has not been characterized due a lack of specific reagents. To address this, we generated a hybridoma (G9P3-1) secreting a monoclonal antibody (G9P3-1Mab), reactive against both human and mouse EVA1. The G9P3-1Mab was generated by immunizing Mpzl2-deficient gene-targeted mice with the extracellular domain of EVA1, followed by a conventional hybridoma fusion protocol, illustrating the feasibility of using gene-targeted mice to generate monoclonal antibodies with multiple species cross-reactivity. We confirmed expression of EVA1 on cortical and medullary epithelial cell subsets and revealed a restricted pattern of expression on CD4- CD8- double negative (DN) cell subsets, with the highest level of expression on DN3 (CD44lowCD25+) thymocytes. G9P3-1MAb is a valuable reagent to study thymic T cell development and is likely useful for the analysis of pathological conditions affecting thymopoiesis, such as thymic involution caused by stress or aging. Fil: Garabatos, Nahir. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Blanco, Jesus. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Fandos, Cesar. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Lopez, Elena. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Santamaria, Pere. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España. University of Calgary; Canadá Fil: Ruiz, Andrea. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España Fil: Perez Vidakovics, Maria Laura Anabella. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Benveniste, Patricia. University of Toronto; Canadá Fil: Galkin, Oleksandr. University of Toronto; Canadá Fil: Zuñiga Pflucker, Juan Carlos. University of Toronto; Canadá Fil: Serra, Pau. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España

Regulation of gene expression in response to local iron concentration is commonly observed in bacterial pathogens that face this nutrient limitation during host infection. In this study, a proteomic approach was used to analyze the differential protein expression of Bordetella pertussis under iron limitation. Whole cell lysates (WCL) and outer membrane fractions of bacteria grown either under iron-starvation or iron-excess conditions were analyzed by two-dimensional (2-D) gel electrophoresis. Statistical analysis revealed 36 proteins displaying differential expression, 9 with higher expression under iron-excess and 27 with increased expression under iron-starvation. These proteins were subjected to tryptic digestion and MALDI-TOF MS. Apart from those previously reported, we identified new low-iron-induced proteins that might help to explain the increased virulence of this phenotype. Additionally, we found evidence that at least one of the identified proteins, solely expressed under iron starvation, is highly immunogenic in infected individuals.

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