Formulated Phospholipids as Non-Canonical TLR4 Agonists.

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.

Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19+ subjects.

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

Development of a VRC01-class germline targeting immunogen derived from anti-idiotypic antibodies.

An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs). Broad and potent VRC01-class bNAbs have been isolated from multiple infected individuals, suggesting that they could be reproducibly elicited by vaccination. Several HIV-1 envelope-derived germline-targeting immunogens have been designed to engage naive VRC01-class precursor B cells. However, they also present off-target epitopes that could hinder development of VRC01-class bNAbs. We characterize a panel of anti-idiotypic monoclonal antibodies (ai-mAbs) raised against inferred-germline (iGL) VRC01-class antibodies. By leveraging binding, structural, and B cell sorting data, we engineered a bispecific molecule derived from two ai-mAbs; one specific for VRC01-class heavy chains and one specific for VRC01-class light chains. The bispecific molecule preferentially activates iGL-VRC01 B cells in vitro and induces specific antibody responses in a murine adoptive transfer model with a diverse polyclonal B cell repertoire. This molecule represents an alternative non-envelope-derived germline-targeting immunogen that can selectively activate VRC01-class precursors in vivo.

A single mRNA immunization boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection.

Emerging SARS-CoV-2 variants have raised concerns about resistance to neutralizing antibodies elicited by previous infection or vaccination. We examined whether sera from recovered and naive donors collected prior to, and following immunizations with existing mRNA vaccines, could neutralize the Wuhan-Hu-1 and B.1.351 variants. Pre-vaccination sera from recovered donors neutralized Wuhan-Hu-1 and sporadically neutralized B.1.351, but a single immunization boosted neutralizing titers against all variants and SARS-CoV-1 by up to 1000-fold. Neutralization was due to antibodies targeting the receptor binding domain and was not boosted by a second immunization. Immunization of naïve donors also elicited cross-neutralizing responses, but at lower titers. Our study highlights the importance of vaccinating both uninfected and previously infected persons to elicit cross-variant neutralizing antibodies.

mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection.

Emerging SARS-CoV-2 variants have raised concerns about resistance to neutralizing antibodies elicited by previous infection or vaccination. We examined whether sera from recovered and naïve donors collected prior to, and following immunizations with existing mRNA vaccines, could neutralize the Wuhan-Hu-1 and B.1.351 variants. Pre-vaccination sera from recovered donors neutralized Wuhan-Hu-1 and sporadically neutralized B.1.351, but a single immunization boosted neutralizing titers against all variants and SARS-CoV-1 by up to 1000-fold. Neutralization was due to antibodies targeting the receptor binding domain and was not boosted by a second immunization. Immunization of naïve donors also elicited cross-neutralizing responses, but at lower titers. Our study highlights the importance of vaccinating both uninfected and previously infected persons to elicit cross-variant neutralizing antibodies.

Development and Testing of a Spray-Dried Tuberculosis Vaccine Candidate in a Mouse Model.

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.

Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation.

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.

Preclinical optimization of an enterotoxigenic Escherichia coli adjuvanted subunit vaccine using response surface design of experiments.

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.

Characterization of neutralizing antibodies from a SARS-CoV-2 infected individual.

B cells specific for the SARS-CoV-2 S envelope glycoprotein spike were isolated from a COVID-19-infected subject using a stabilized spike-derived ectodomain (S2P) twenty-one days post-infection. Forty-four S2P-specific monoclonal antibodies were generated, three of which bound to the receptor binding domain (RBD). The antibodies were minimally mutated from germline and were derived from different B cell lineages. Only two antibodies displayed neutralizing activity against SARS-CoV-2 pseudo-virus. The most potent antibody bound the RBD in a manner that prevented binding to the ACE2 receptor, while the other bound outside the RBD. Our study indicates that the majority of antibodies against the viral envelope spike that were generated during the first weeks of COVID-19 infection are non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 spike-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive/therapeutic potential and can serve as templates for vaccine-design.

Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation.

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.

Selective inhibition of anti-MAG IgM autoantibody binding to myelin by an antigen-specific glycopolymer.

Anti-myelin-associated glycoprotein (MAG) neuropathy is a disabling autoimmune peripheral neuropathy that is caused by circulating monoclonal IgM autoantibodies directed against the human natural killer-1 (HNK-1) epitope. This carbohydrate epitope is highly expressed on adhesion molecules such as MAG, a glycoprotein present in myelinated nerves. We previously showed the therapeutic potential of the glycopolymer poly(phenyl disodium 3-O-sulfo-ß-d-glucopyranuronate)-(1→3)-ß-d-galactopyranoside (PPSGG) in selectively neutralizing anti-MAG IgM antibodies in an immunological mouse model and ex vivo with sera from anti-MAG neuropathy patients. PPSGG is composed of a biodegradable backbone that multivalently presents a mimetic of the HNK-1 epitope. In this study, we further explored the pharmacodynamic properties of the glycopolymer and its ability to inhibit the binding of anti-MAG IgM to peripheral nerves. The polymer selectively bound anti-MAG IgM autoantibodies and prevented the binding of patients' anti-MAG IgM antibodies to myelin of non-human primate sciatic nerves. Upon PPSGG treatment, neither activation nor inhibition of human and murine peripheral blood mononuclear cells nor alteration of systemic inflammatory markers was observed in mice or ex vivo in human peripheral blood mononuclear cells. Intravenous injections of PPSGG to mice immunized against the HNK-1 epitope removed anti-MAG IgM antibodies within less than 1 hr, indicating a fast and efficient mechanism of action as compared to a B-cell depletion with anti-CD20. In conclusion, these observations corroborate the therapeutic potential of PPSGG for an antigen-specific treatment of anti-MAG neuropathy. Read the Editorial Highlight for this article on page 465.

The TLR4 agonist adjuvant SLA-SE promotes functional mucosal antibodies against a parenterally delivered ETEC vaccine.

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.

Reprogramming the adjuvant properties of aluminum oxyhydroxide with nanoparticle technology.

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.

Effective Combination Adjuvants Engage Both TLR and Inflammasome Pathways To Promote Potent Adaptive Immune Responses.

The involvement of innate receptors that recognize pathogen- and danger-associated molecular patterns is critical to programming an effective adaptive immune response to vaccination. The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) synergizes with the squalene oil-in-water emulsion (SE) formulation to induce strong adaptive responses. Although TLR4 signaling through MyD88 and TIR domain-containing adapter inducing IFN-ß are essential for GLA-SE activity, the mechanisms underlying the synergistic activity of GLA and SE are not fully understood. In this article, we demonstrate that the inflammasome activation and the subsequent release of IL-1ß are central effectors of the action of GLA-SE, as infiltration of innate cells into the draining lymph nodes and production of IFN-γ are reduced in ASC-/- animals. Importantly, the early proliferation of Ag-specific CD4+ T cells was completely ablated after immunization in ASC-/- animals. Moreover, numbers of Ag-specific CD4+ T and B cells as well as production of IFN-γ, TNF-α, and IL-2 and Ab titers were considerably reduced in ASC-/-, NLRP3-/-, and IL-1R-/- mice compared with wild-type mice and were completely ablated in TLR4-/- animals. Also, extracellular ATP, a known trigger of the inflammasome, augments Ag-specific CD4+ T cell responses, as hydrolyzing it with apyrase diminished adaptive responses induced by GLA-SE. These data thus demonstrate that GLA-SE adjuvanticity acts through TLR4 signaling and NLRP3 inflammasome activation to promote robust Th1 and B cell responses to vaccine Ags. The findings suggest that engagement of both TLR and inflammasome activators may be a general paradigm for induction of robust CD4 T cell immunity with combination adjuvants such as GLA-SE.

Virosome-bound antigen enhances DC-dependent specific CD4+ T cell stimulation, inducing a Th1 and Treg profile in vitro.

There is considerable interest to develop antigen-carriers for immune-modulatory clinical applications, but insufficient information is available on their effects on antigen-presenting cells. We employed virosomes coupled to ovalbumin (OVA) to study their interaction with murine bone marrow-derived dendritic cells (BMDCs) and modulation of downstream T cell responses. BMDCs were treated in vitro with virosomes or liposomes prior to determining BMDC phenotype, viability, and intracellular trafficking. Antigen-specific CD4+ T cell activation was measured by co-culture of BMDCs with DO11.10 CD4+ T cells. Compared to liposomes, virosomes were rapidly taken up. Neither nanocarrier type affected BMDC viability, nor did a moderate degree of activation differ for markers such as CD40, CD80, CD86. Virosome uptake occurred via clathrin-mediated endocytosis and phagocytosis, with co-localization in late endosomes. Only BMDCs treated with OVA-coupled virosomes induced enhanced OVA-specific CD4+ T cell proliferation. Antigen-coupled virosomes are endowed with an intrinsic ability to modulate DC-dependent adaptive immune responses.

Interaction of biomedical nanoparticles with the pulmonary immune system.

Engineered nanoparticles (NPs) offer site-specific delivery, deposition and cellular uptake due to their unique physicochemical properties and were shown to modulate immune responses. The respiratory tract with its vast surface area is an attractive target organ for innovative immunomodulatory therapeutic applications by pulmonary administration of such NPs, enabling interactions with resident antigen-presenting cells (APCs), such as dendritic cells and macrophages. Depending on the respiratory tract compartment, e.g. conducting airways, lung parenchyma, or lung draining lymph nodes, APCs extensively vary in their number, morphology, phenotype, and function. Unique characteristics and plasticity render APC populations ideal targets for inhaled specific immunomodulators. Modulation of immune responses may operate in different steps of the immune cell-antigen interaction, i.e. antigen uptake, trafficking, processing, and presentation to T cells. Meticulous analysis of the immunomodulatory potential, as well as pharmacologic and biocompatibility testing of inhalable NPs is required to develop novel strategies for the treatment of respiratory disorders such as allergic asthma. The safe-by-design and characterization of such NPs requires well coordinated interdisciplinary research uniting engineers, chemists biologists and respiratory physicians. In this review we will focus on in vivo data available to facilitate the design of nanocarrier-based strategies using NPs to modulate pulmonary immune responses.

Current in vitro approaches to assess nanoparticle interactions with lung cells.

The respiratory tract is in constant contact with inhaled antigens from the external environment. In order to shape its line of defense, it is populated by various types of immune cells. Taking into account the scientific breakthroughs of nanomedicine and nanoparticle drug delivery, we can think of the respiratory tract as an ideal target organ to study and develop nanocarrier-based vaccines to treat respiratory tract disorders. Nanoparticles have been proven capable of specific cell targeting and, when suitably engineered, are able to induce an immunomodulatory effect. The aim of this review is to highlight in vitro approaches to the study of nanoparticle-lung immune cell interactions and recent advances in the targeting of immune cells using nanoparticle-based systems.

Pulmonary delivery of cationic gold nanoparticles boost antigen-specific CD4+ T Cell Proliferation.

To address how surface charge affects the fate of potential nanocarriers in the lung, gold nanoparticles (AuNPs) coated with polyvinyl alcohol containing either positively (NH2) or negatively (COOH) charged functional groups were intra-nasally instilled in mice, and their uptake by antigen presenting cell populations (APC) in broncho-alveolar lavage (BAL) fluid, trachea, and lung parenchyma, as well as trafficking to the lung draining lymph nodes (LDLNs) was assessed by flow cytometry. Furthermore, CD4+ T cell proliferation in LDLNs was investigated following instillation. All APC subpopulations preferentially captured positively-charged AuNPs compared to their negatively-charged counterparts. Uptake of AuNPs up-regulated expression of co-stimulatory molecules on all APC populations. Furthermore, positively-charged AuNPs induced enhanced OVA-specific CD4+ T cell stimulation in LDLNs compared to negatively-charged AuNPs, or polymer alone. Our findings demonstrate surface charge as a key parameter determining particle uptake by APC, and down-stream immune responses depend on the presence of particle core-bound polymer.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS: Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS: The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.