Lipid Nanoparticle Encapsulation Empowers Poly(I:C) to Activate Cytoplasmic RLRs and Thereby Increases Its Adjuvanticity.

Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.

Visible Light Conjugation with Triazolinediones as a Route to Degradable Poly(ethylene glycol)-Lipids for mRNA Lipid Nanoparticle Formulation.

Polyethylene glycol (PEG) is considered as the gold standard for colloidal stabilization of nanomedicines, yet PEG is non-degradable and lacks functionality on the backbone. Herein, we introduce concomitantly PEG backbone functionality and degradability via a one-step modification with 1,2,4-triazoline-3,5-diones (TAD) under green light. The TAD-PEG conjugates are degradable in aqueous medium under physiological conditions, with the rate of hydrolysis depending on pH and temperature. Subsequently, a PEG-lipid is modified with TAD-derivatives and successfully used for messenger RNA (mRNA) lipid nanoparticle (LNP) delivery, thereby improving mRNA transfection efficiency on multiple cell cultures in vitro. In vivo, in mice, mRNA LNP formulation exhibited a similar tissue distribution as common LNPs, with a slight decrease in transfection efficiency. Our findings pave the road towards the design of degradable, backbone-functionalized PEG for applications in nanomedicine and beyond.

Corticosteroids and cellulose purification improve, respectively, the in vivo translation and vaccination efficacy of sa-mRNAs.

Synthetic mRNAs are an appealing platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison with conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining interest because of their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate their clinical application. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. Here we investigated, in vivo, the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response and the translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that corticosteroids and especially topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented formation of antibodies against sa-mRNA-encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate a drastic reduction of these dsRNA by-products upon cellulose-based purification, reducing the innate immune response and improving sa-mRNA vaccination efficacy.

Squaric Ester-Based, pH-Degradable Nanogels: Modular Nanocarriers for Safe, Systemic Administration of Toll-like Receptor 7/8 Agonistic Immune Modulators.

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine*.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.

Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

Poly(2-ethyl-2-oxazoline) Conjugates with Salicylic Acid via Degradable Modular Ester Linkages.

Conjugation of drugs to polymers is a widely used approach to gain control over the release of therapeutics. In this contribution, salicylic acid, a multipurpose model drug, is conjugated to the biocompatible poly(2-ethyl-2-oxazoline) (PEtOx). The drug is attached to the side chains of a polymer carrier through a hydrolytically cleavable ester linker, via a sequential postpolymerization modification. The chemical modulation of this ester, i.e., by primary or secondary alcohols, is demonstrated to greatly influence the ester hydrolysis rate. This crucial parameter allows us to tune the in vitro kinetics of the sustained drug release for periods exceeding a month in phosphate-buffered saline (PBS). The synthetic accessibility of the cleavable linker, together with the modularity of the drug release rate offered by this approach, highlights the utility of this class of polymers in the field of long-lasting drug delivery systems for persistent and chronic disease treatment.

Current Status of COVID-19 (Pre)Clinical Vaccine Development.

The current COVID-19 pandemic has a tremendous impact on daily life world-wide. Despite the ability to dampen the spread of SARS-CoV-2, the causative agent of the diseases, through restrictive interventions, it is believed that only effective vaccines will provide sufficient control over the disease and revert societal live back to normal. At present, a double-digit number of efforts are devoted to the development of a vaccine against COVID-19. Here, we provide an overview of these (pre)clinical efforts and provide background information on the technologies behind these vaccines. In addition, we discuss potential hurdles that need to be addressed prior to mass scale clinical translation of successful vaccine candidates.

Potent Lymphatic Translocation and Spatial Control Over Innate Immune Activation by Polymer-Lipid Amphiphile Conjugates of Small-Molecule TLR7/8 Agonists.

Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid-polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid-polymer amphiphiles for optimized lymphoid targeting.

Lymph-Node-Targeted Immune Activation by Engineered Block Copolymer Amphiphiles-TLR7/8 Agonist Conjugates.

Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.

Exploration of Solid Phase Peptoid Synthesis for the Design of Trifunctional Hapten-Lipid-TLR7/8 Agonist Antibody-Recruiting Oligomers That Combine Innate Effector with Innate Activation Function.

The strategic engagement of innate immunity is a promising avenue for cancer treatment. Antibody-recruiting molecules (ARMs) direct endogenous antibodies to target tumor sites, eliciting innate immune effector killing responses. In this study, we report the synthesis of ARMs by employing solid-phase peptoid synthesis to construct three libraries of antibody-recruiting oligomers. Using dinitrophenyl (DNP) as a model hapten and alkyl lipid chains for cell surface anchoring, we tailored oligomers with variations in valency and spatial configuration. Among these, an oligomer design featuring DNP connected to the oligomer backbone through an extended PEG linker and flanked by two lipid motifs emerged as the most effective in antibody recruitment in vitro. This oligomer was further functionalized to include an imidazoquinoline, creating a trifunctional hapten-lipid-TLR7/8 agonist oligomer, and a parallel variant was conjugated with rhodamine, resulting in a trifunctional hapten-lipid-dye oligomer. Upon intratumorally administration in a murine model, these oligomers induced localized immune activation within tumors. Subsequent ex vivo analysis of single-cell suspensions from excised tumors confirmed the enhanced binding of anti-DNP antibodies. These findings underscore the potential of custom-designed ARMs in orchestrating precise immune-mediated tumor targeting and highlight the adaptability of solid-phase synthesis in oligomer design for the design of multifunctional antibody recruiting molecules.

Immunogenicity and biodistribution of lipid nanoparticle formulated self-amplifying mRNA vaccines against H5 avian influenza.

This study reports on the immunogenicity and biodistribution of H5 hemagglutinin (HA)-based self-amplifying (sa) mRNA vaccines in mice. Four sa-mRNA vaccines encoding either a secreted full-length HA, a secreted HA head domain, a secreted HA stalk domain, or a full-length membrane-anchored HA were investigated. All vaccines elicited an adaptive immune response. However, the full-length HA sa-RNA vaccines demonstrated superior performance compared to head and stalk domain vaccines. The antibody titers positively correlated with the vaccine dose. Cellular immune responses and antigen-specific IgA antibodies in the lungs were also observed. The comparison of the sa-mRNA vaccines encoding the secreted and membrane-anchored full-length HA revealed that anchoring of the HA to the membrane significantly enhanced the antibody and cellular responses. In addition to the injection site, the intramuscularly injected sa-mRNA-LNPs were also detected in the draining lymph nodes, spleen, and to a lesser extent, in the lung, kidney, liver, and heart.

PH-Triggered, Lymph Node Focused Immunodrug Release by Polymeric 2-Propionic-3-Methyl-maleic Anhydrides with Cholesteryl End Groups.

Gaining spatial control over innate immune activation is of great relevance during vaccine delivery and anticancer therapy, where one aims at activating immune cells at draining lymphoid tissue while avoiding systemic off-target innate immune activation. Lipid-polymer amphiphiles show high tendency to drain to lymphoid tissue upon local administration. Here, pH-sensitive, cholesteryl end group functionalized polymers as stimuli-responsive carriers are introduced for controlled immunoactivation of draining lymph nodes. Methacrylamide-based monomers bearing pendant 2-propionic-3-methylmaleic anhydride groups are polymerized by Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization using a cholesterol chain-transfer agent (chol-CTA). The amine-reactive anhydrides are conjugated with various amines, however, while primary amines afforded irreversible imides, secondary amines provided pH-responsive conjugates that are released upon acidification. This can be applied to fluorescent dyes for irreversibly carrier labeling or immunostimulatory Toll-like receptor (TLR) 7/8 agonists as cargos for pH-responsive delivery. Hydrophilization of remaining anhydride repeating units with short PEG-chains yielded cholesteryl-polymer amphiphiles that showed efficient cellular uptake and increased drug release at endosomal pH. Moreover, reversibly conjugated TLR 7/8 agonist amphiphiles efficiently drained to lymph nodes and increased the number of effectively maturated antigen-presenting cells after subcutaneous injection in vivo. Consequently, cholesteryl-linked methacrylamide-based polymers with pH-sensitive 2-propionic-3-methylmaleic anhydride side groups provide ideal features for immunodrug delivery.

Amplification of Protein Expression by Self-Amplifying mRNA Delivered in Lipid Nanoparticles Containing a ß-Aminoester Ionizable Lipid Correlates with Reduced Innate Immune Activation.

Self-amplifying mRNA (saRNA) is witnessing increased interest as a platform technology for protein replacement therapy, gene editing, immunotherapy, and vaccination. saRNA can replicate itself inside cells, leading to a higher and more sustained production of the desired protein at a lower dose. Controlling innate immune activation, however, is crucial to suppress unwanted inflammation upon delivery and self-replication of RNA in vivo. In this study, we report on a class of ß-aminoester lipids (ßAELs) synthesized through the Michael addition of an acrylate to diethanolamine, followed by esterification with fatty acids. These lipids possessed one or two ionizable amines, depending on the use of nonionic or amine-containing acrylates. We utilized ßAELs for encapsulating saRNA in lipid nanoparticles (LNPs) and evaluated their transfection efficiency in vitro and in vivo in mice, while comparing them to LNPs containing ALC-0315 as an ionizable lipid reference. Among the tested lipids, OC7, which comprises two unsaturated oleoyl alkyl chains and an ionizable azepanyl motif, emerged as a ßAEL with low cytotoxicity and immunogenicity relative to ALC-0315. Interestingly, saRNA delivered via the OC7 LNP exhibited a distinct in vivo transfection profile. Initially, intramuscular injection of OC7 LNP resulted in low protein expression shortly after administration, followed by a gradual increase over a period of up to 7 days. This pattern is indicative of successful self-amplification of saRNA. In contrast, saRNA delivered via ALC-0315 LNP demonstrated high protein translation initially, which gradually declined over time and lacked the amplification seen with OC7 LNP. We observed that, in contrast to saRNA OC7 LNP, saRNA ALC-0315 LNP induced potent innate immune activation by triggering cytoplasmic RIG-I-like receptors (RLRs), likely due to the highly efficient endosomal membrane rupturing properties of ALC-0315 LNP. Consequently, the massive production of type I interferons quickly hindered the amplification of the saRNA. Our findings highlight the critical role of the choice of ionizable lipid for saRNA formulation in LNPs, particularly in shaping the qualitative profile of protein expression. For applications where minimizing inflammation is desired, the use of ionizable lipids, such as the ßAEL reported in this study, that elicit a low type I interferon response in saRNA LNP is crucial.

CO-DELIVERY of glutamic acid-extended peptide antigen and imidazoquinoline TLR7/8 agonist via ionizable lipid nanoparticles induces protective anti-tumor immunity.

Cancer vaccines aim at generating cytotoxic CD8+ T cells that kill cancer cells and confer durable tumor regression. Hereto, CD8+ peptide epitopes should be presented by antigen presenting cells to CD8+ T cells in lymphoid tissue. Unfortunately, in unformulated soluble form, peptide antigens are poorly taken up by antigen presenting cells and do not efficiently reach lymph nodes. Hence, the lack of efficient delivery remains a major limitation for successful clinical translation of cancer vaccination using peptide antigens. Here we propose a generic peptide nanoformulation strategy by extending the amino acid sequence of the peptide antigen epitope with 10 glutamic acid residues. The resulting overall anionic charge of the peptide allows encapsulation into lipid nanoparticles (peptide-LNP) by electrostatic interaction with an ionizable cationic lipid. We demonstrate that intravenous injection of peptide-LNP efficiently delivers the peptide to immune cells in the spleen. Peptide-LNP that co-encapsulate an imidazoquinoline TLR7/8 agonist (IMDQ) induce robust innate immune activation in a broad range of immune cell subsets in the spleen. Peptide-LNP containing the minimal CD8+ T cell epitope of the HPV type 16 E7 oncoprotein and IMDQ induces high levels of antigen-specific CD8+ T cells in the blood, and can confer protective immunity against E7-expressing tumors in both prophylactic and therapeutic settings.

Successful batch and continuous lyophilization of mRNA LNP formulations depend on cryoprotectants and ionizable lipids.

The limited thermostability and need for ultracold storage conditions are the major drawbacks of the currently used nucleoside-modified lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccines, which hamper the distribution of these vaccines in low-resource regions. The LNP core contains, besides mRNA and lipids, a large fraction of water. Therefore, encapsulated mRNA, or at least a part of it, is subjected to hydrolysis mechanisms similar to unformulated mRNA in an aqueous solution. It is likely that the hydrolysis of mRNA and colloidal destabilization are critical factors that decrease the biological activity of mRNA LNPs upon storage under ambient conditions. Hence, lyophilization as a drying technique is a logical and appealing method to improve the thermostability of these vaccines. In this study, we demonstrate that mRNA LNP formulations comprising a reduction-sensitive ionizable lipid can be successfully lyophilized, in the presence of 20% w/v sucrose, both by conventional batch freeze-drying and by an innovative continuous spin lyophilization process. While the chemical structure of the ionizable lipid did not affect the colloidal stability of the LNP after lyophilization and redispersion in an aqueous medium, we found that the ability of LNPs to retain the mRNA payload stably encapsulated, and mediate in vivo and in vitro mRNA translation into protein, post lyophilization strongly depended on the ionizable lipid in the LNP formulation.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Lipid-Polyglutamate Nanoparticle Vaccine Platform.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(l-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.