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SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determine the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain. The structure reveals that CV30 binds to an epitope that overlaps with the human ACE2 receptor binding motif providing a structural basis for its neutralization. CV30 also induces shedding of the S1 subunit, indicating an additional mechanism of neutralization. A germline reversion of CV30 results in a substantial reduction in both binding affinity and neutralization potential indicating the minimal somatic mutation is needed for potently neutralizing antibodies against SARS-CoV-2. Currently there is neither a vaccine nor an effective treatment strategy available for COVID19. Here, Hurlburt et al. provide the crystal structure of a patient-derived monoclonal antibody neutralizing SARS-CoV-2 via shedding of the S1 subunit and competing for the receptor binding domain.

Cultures of primary human nasal epithelial cells (hNECs) differentiated at the air–liquid interface (ALI) represent a sophisticated and widely used model of the human upper respiratory epithelium. Despite the availability of various cell culture insert types and the well-established understanding that different culture media influence the cell culture characteristics, the possible impact of the insert brand remains rather underexplored. We cultured hNECs from nineteen healthy adult donors on three distinct brands of commercially available inserts—Corning® Transwell®, CELLTREAT®, and ThinCert®—and compared the ciliary activity and cellular composition of the cultures using high-speed video microscopy and flow cytometry, respectively. Additionally, we employed an alternative method of hNEC culture setup—the inverted condition—wherein the hNECs were seeded on the basal side of the insert with the idea to avoid mucus accumulation. Our results show that ciliary activity and cell type composition did not differ between insert types for both culture conditions. However, we found a higher ciliary beat frequency and a lower active (ciliated) area in the inverted setup compared to the conventional setup across all three insert brands. These findings indicate that all three mentioned insert types yield comparable cell cultures.

Aluminum salts, developed almost a century ago, remain the most commonly used adjuvant for licensed human vaccines. Compared to more recently developed vaccine adjuvants, aluminum adjuvants such as Alhydrogel are heterogeneous in nature, consisting of 1–10 micrometer-sized aggregates of nanoparticle aluminum oxyhydroxide fibers. To determine whether the particle size and aggregated state of aluminum oxyhydroxide affects its adjuvant activity, we developed a scalable, top-down process to produce stable nanoparticles (nanoalum) from the clinical adjuvant Alhydrogel by including poly(acrylic acid) (PAA) polymer as a stabilizing agent. Surprisingly, the PAA:nanoalum adjuvant elicited a robust TH1 immune response characterized by antigen-specific CD4 + T cells expressing IFN-γ and TNF, as well as high IgG2 titers, whereas the parent Alhydrogel and PAA elicited modest TH2 immunity characterized by IgG1 antibodies. ASC, NLRP3 and the IL-18R were all essential for TH1 induction, indicating an essential role of the inflammasome in this adjuvant’s activity. Compared to microparticle Alhydrogel this nanoalum adjuvant provided superior immunogenicity and increased protective efficacy against lethal influenza challenge. Therefore PAA:nanoalum represents a new class of alum adjuvant that preferentially enhances TH1 immunity to vaccine antigens. This adjuvant may be widely beneficial to vaccines for which TH1 immunity is important, including tuberculosis, pertussis, and malaria. Adjuvants: Nanoalum gives a boost Aluminum salt-based adjuvants such as alhydrogel have been a mainstay of vaccines for decades. Christopher B. Fox and colleagues at the Infectious Disease Research Institute in Seattle, USA, investigate the effect adjuvant particle size has on experimental vaccine responses. Shearing conventional micrometer-scale alhyrodogel into nanoparticles is followed by rapid reaggregation; however, the authors show that addition of anionic polymer (PAA) prevents this and results in stable nanoparticles (PAA:nanoalum). Used as an adjuvant with either influenza or TB antigens triggers robust TH1 and IgG2a responses that are superior to alhydrogel even when the latter includes a Toll-like receptor 4 (TLR4) agonist. Interestingly, addition of TLR4 agonist to PAA:nanoalum actually impairs its adjuvanticity. PAA:nanoalum efficacy is nevertheless dependent on the NLRP3 inflammasome suggesting that this novel adjuvant somehow triggers this pathway through some as yet undefined route.

Converting a vaccine into a thermostable dry powder is advantageous as it reduces the resource burden linked with the cold chain and provides flexibility in dosage and administration through different routes. Such a dry powder presentation may be especially useful in the development of a vaccine towards the respiratory infectious disease tuberculosis (TB). This study assesses the immunogenicity and protective efficacy of spray-dried ID93+GLA-SE, a promising TB vaccine candidate, against Mycobacterium tuberculosis (Mtb) in a murine model when administered via different routes. Four administration routes for the spray-dried ID93+GLA-SE were evaluated along with relevant controls—1) reconstitution and intramuscular injection, 2) reconstitution and intranasal delivery, 3) nasal dry powder delivery via inhalation, and 4) pulmonary dry powder delivery via inhalation. Dry powder intranasal and pulmonary delivery was achieved using a custom nose-only inhalation device, and optimization using representative vaccine-free powder demonstrated that approximately 10 and 44% of the maximum possible delivered dose would be delivered for intranasal delivery and pulmonary delivery, respectively. Spray-dried powder was engineered according to the different administration routes including maintaining approximately equivalent delivered doses of ID93 and GLA. Vaccine properties of the different spray-dried lots were assessed for quality control in terms of nanoemulsion droplet diameter, polydispersity index, adjuvant content, and antigen content. Our results using the Mtb mouse challenge model show that both intranasal reconstituted vaccine delivery as well as pulmonary dry powder vaccine delivery resulted in Mtb control in infected mice comparable to traditional intramuscular delivery. Improved protection in these two vaccinated groups over their respective control groups coincided with the presence of cytokine-producing T cell responses. In summary, our results provide novel vaccine formulations and delivery routes that can be harnessed to provide protection against Mtb infection.

VRC01-class antibodies are a genetically restricted class of antibodies capable of potently neutralizing diverse strains of HIV-1. Unmutated VRC01 precursors fail to recognize recombinant HIV-1 Envelope (Env) proteins, which necessitated the development of germline targeting vaccine immunogens capable of initiating VRC01-class B cell response. Among these, we developed an anti-idiotypic monoclonal antibody (ai-mAb)-derived VRC01 class targeting immunogen. Because it is distinct from Env, we speculated that the ai-mAb will selectively engage naive VRC01 class B cells while limiting B cell responses directed at off-target epitopes on Env during prime-boost regimens. Here, we evaluated the serum and B cell responses to ai-mAb prime/Env boost, and Env-prime/Env boost regimens in a murine adoptive transfer model where VRC01 precursor B cells are present at physiological levels. We found that the Env-Env regimen led to the greatest expansion of on-target VRC01 B cells, drove larger VRC01-class GC responses, and elicited higher titers of circulating antibodies despite also eliciting substantial off-target Env-specific responses. Single-cell sorting experiments revealed that the ai-mAb was driving off-track somatic mutations. IgG transfer experiments demonstrated that circulating off-target antibodies provide a positive feedback mechanism that potentiates on-target B cell responses. Collectively, the results suggest that non-Env immunogens are not ideal for priming VRC01-class B cells, where sequential boosting with Env will be required to drive maturation of neutralizing breadth.

Immunogenic agents known as adjuvants play a critical role in many vaccine formulations. Adjuvants often signal through Toll-like receptor (TLR) pathways, including formulations in licensed vaccines that target TLR4. While TLR4 is predominantly known for responding to lipopolysaccharide (LPS), a component of Gram-negative bacterial membranes, it has been shown to be a receptor for a number of molecular structures, including phospholipids. Therefore, phospholipid-based pharmaceutical formulations might have off-target effects by signaling through TLR4, confounding interpretation of pharmaceutical bioactivity. In this study we examined the individual components of a clinical stage oil-in-water vaccine adjuvant emulsion (referred to as a stable emulsion or SE) and their ability to signal through murine and human TLR4s. We found that the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) activated TLR4 and elicited many of the same immune phenotypes as canonical TLR4 agonists. This pathway was dependent on the saturation, size, and headgroup of the phospholipid. Interestingly, DMPC effects on human cells were evident but overall appeared less impactful than emulsion oil composition. Considering the prevalence of DMPC and other phospholipids used across the pharmaceutical space, these findings may contextualize off-target innate immune responses that could impact preclinical and clinical development.

Many pathogens establish infection at mucosal surfaces such as the enteric pathogen Enterotoxigenic E. coli (ETEC). Thus, there is a pressing need for effective vaccination strategies that promote protective immunity at mucosal surfaces. Toll-like receptor (TLR) ligands have been extensively developed as vaccine adjuvants to promote systemic immunity, whereas attenuated bacterial toxins including cholera toxin and heat-labile toxin (LT) have initially been developed to promote mucosal immunity. Here we evaluate the ability of the TLR4 agonist second-generation lipid adjuvant formulated in a stable emulsion (SLA-SE) to augment functional mucosal antibodies elicited by intramuscular immunization with a recombinant ETEC vaccine antigen. We find that, in mice, parenterally delivered SLA-SE is at least as effective as the double-mutant LT (LTR192G/L211A, dmLT) adjuvant in promoting functional antibodies and eliciting intestinal IgA responses to the vaccine antigen. In addition, SLA-SE enhanced both the IgG2a response in the mucosa and serum, and the production of LT neutralizing serum antibodies elicited by dmLT four to eightfold. These results reveal unexpected mucosal adjuvant properties of this TLR4 agonist adjuvant when delivered intramuscularly. This may have a substantial impact on the development of vaccines against enteric and other mucosal pathogens. Mucosal immunity: parenteral vaccination hits the gut Although offering great potential for generating intestinal immunity, vaccination by the oral route suffers from several barriers such as the breakdown of protein vaccines in the stomach and/or the induction of oral tolerance. To investigate whether these barriers can be circumvented, Mark T. Orr and colleagues at the Infectious Disease Research Institute use a parenteral (intramuscular) vaccination protocol in mice. Intramuscular immunization with an enterotoxigenic E. coli (ETEC) vaccine plus a Toll-like receptor 4 adjuvant in stable emulsion (SLA-SE) elicits a functional antibody response in both the gut and serum. Importantly, this intramuscular vaccination triggers robust production of IgA in the gut. These findings suggest that with the right adjuvant combination it might possible to generate potent protective mucosal immunity following parenteral immunization.

Enterotoxigenic E. coli (ETEC) is a leading cause of moderate-to-severe diarrhoea. ETEC colonizes the intestine through fimbrial tip adhesin colonization factors and produces heat-stable and/or heat-labile (LT) toxins, stimulating fluid and electrolyte release leading to watery diarrhoea. We reported that a vaccine containing recombinant colonization factor antigen (CfaEB) targeting fimbrial tip adhesin of the colonization factor antigen I (CFA/I) and an attenuated LT toxoid (dmLT) elicited mucosal and systemic immune responses against both targets. Additionally, the toll-like receptor 4 ligand second-generation lipid adjuvant (TLR4-SLA) induced a potent mucosal response, dependent on adjuvant formulation. However, a combination of vaccine components at their respective individual optimal doses may not achieve the optimal immune profile. We studied a subunit ETEC vaccine prototype in mice using a response surface design of experiments (DoE), consisting of 64 vaccine dose-combinations of CfaEB, dmLT and SLA in four formulations (aqueous, aluminium oxyhydroxide, squalene-in-water stable nanoemulsion [SE] or liposomes containing the saponin Quillaja saponaria-21 [LSQ]). Nine readouts focusing on antibody functionality and plasma cell response were selected to profile the immune response of parenterally administered ETEC vaccine prototype. The data were integrated in a model to identify the optimal dosage of each vaccine component and best formulation. Compared to maximal doses used in mouse models (10 µg CfaEB, 1 µg dmLT and 5 µg SLA), a reduction in the vaccine components up to 37%, 60% and 88% for CfaEB, dmLT and SLA, respectively, maintained or even maximized immune responses, with SE and LSQ the best formulations. The DoE approach can help determine the best vaccine composition with a limited number of experiments and may accelerate development of multi-antigen/component ETEC vaccines.

The respiratory tract with its vast surface area and very thin air-blood tissue barrier presents an extremely large interface for potential interaction with xenobiotics such as inhaled pathogens or medicaments. To protect its large and vulnerable surface, the lung is populated with several different types of immune cells. Pulmonary epithelial cells, macrophages and dendritic cells are key players in shaping the innate and adaptive immune response. Due to their localization, they represent a frontline of cell populations that are among the first to come in contact with inhaled xenobiotics. Furthermore, depending on the lung compartment they populate, these cells show a large variety in morphology, phenotype, and function. These unique characteristics make those cell populations ideal targets for specific immunomodulators that are designed for inhalation. Depending on cell population or lung compartment targeting, a specific immune response may be triggered or modulated. The purpose of a potent carrier for pulmonary immunomodulation is, first, to efficiently target a specific immunocompetent cell and, second, to affect its role in generating an immune response. Immunomodulation may occur at different levels of immune cell-antigen interaction, i.e. antigen uptake, trafficking, processing and presentation. Inhalation of nanosized carriers for drugs or vaccines shows great potential for both prophylactic and therapeutic approaches in order to modulate immune responses locally or systemically, due to the specific deposition and targeting properties of nanoparticles. Immune responses triggered by nanosized particles may be either immunostimulatory or immunosuppressive and depending on the specific purpose, stimulation or suppression may either be desired or unwanted. Meticulous analysis of immunomodulatory potential, pharmacologic and toxicologic testing of inhalable nanocarriers is required in order to find novel and optimal approaches for prophylaxis and therapy of pulmonary diseases. The design and characterization of such nanoparticles requires well-coordinated interdisciplinary research among engineers, biologists and clinicians.

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3⁺ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4⁺ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.