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Three decades of messenger RNA vaccine development
Abstract and Figures
In the early nineties, pioneering steps were taken in the use of mRNA as a therapeutic tool for vaccination. In the following decades, an improved understanding of the mRNA pharmacology, together with novel insights in immunology have positioned mRNA-based technologies as next-generation vaccines. This review outlines the history and current state-of-the-art in mRNA vaccination, while presenting an immunological view on mRNA vaccine development. As such, we highlight the challenges in vaccine design, testing and administration, key considerations in the design of mRNA-based vaccines and new opportunities that arise when packaging mRNA in nanoparticulate vaccines. Finally, we discuss the mRNA self-adjuvant effect as a critical, but dichotomous parameter that determines the safety, efficacy and strength of the evoked immune response.
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... Lipid nanoparticles (LNPs), a common vehicle for delivering mRNA into cells, can also contribute to inflammatory responses. Studies have shown that certain components of LNPs, such as polyethylene glycol (PEG), may elicit allergic reactions in some individuals [57]. Additionally, the inflammatory pathways activated by mRNA and its delivery systems may lead to systemic reactions if not properly regulated. ...
... For instance, excessive inflammation in vital organs like the liver or kidneys could impair their function, resulting in toxicity that might not be immediately apparent but could manifest after months or years of treatment. Long-term monitoring of patients receiving mRNA therapies is crucial to identify and mitigate these risks [57,58]. ...
Messenger RNA (mRNA) vaccines have quickly become a potent tool in cancer immunotherapy, with the potential to personalise cancer treatment and get around many of the drawbacks of conventional therapies. With an emphasis on their processes, technological developments, and clinical applications, this study examines the scientific breakthroughs and difficulties surrounding mRNA cancer vaccines. We begin by investigating the ways in which mRNA vaccines work to stimulate the immune system, encode antigens specific to tumours, and elicit a potent anti-tumor response. We also look at the variety of mRNA vaccines which are currently being deployed and emphasize their successes in clinical studies, especially when combined with immune checkpoint inhibitors. The effectiveness and safety of these vaccines have been significantly increased by technological developments, such as enhancements in mRNA stability, design, and delivery systems (such as lipid nanoparticles and polyplexes). Emerging technologies like circular RNA and self-amplifying present promising opportunities for enduring and more potent therapies. But there are still limitations with mRNA vaccines, such as stability, immunogenicity, degradation, and effective in vivo delivery. Concerns about possible adverse effects and safety in long-term applications are also covered in this review. Despite these obstacles, novel approaches are being developed to boost antigen expression, optimize delivery systems, and improve mRNA stability, establishing mRNA vaccines as a crucial component of tailored cancer immunotherapy. These developments provide the foundation for upcoming advances in mRNA vaccine technology and represent a major breakthrough in the fight against cancer.
... Future thinking by researchers helps a government build resilient strategies and evaluate the success of existing strategies. Wong and Bartlett's research [1], our own critique, and that of previous researchers [43] might impact policy directly or might need to be 'translated' by others, such as people in the media, into a more understandable format which is more easily absorbed by influential decision-makers including those in government and elites who may champion the cause [44,45]. ...
... Some scientists claim that the discovery of dendritic cells in 1961 can be considered the earliest pioneering milestone of mRNA-based vaccines. Thereafter, rigorous scientific research led to the discovery of critical knowledge in cell biology, which was crucial to the rapid deployment of the COVID-19 mRNA vaccines of contemporary times [45]. Such vaccines, and other academically driven research on pandemics, provided the bed-rock of government responses to the COVID-19 epidemic and the relatively successful mitigation of this singularity. ...
Wong and Bartlett explain the Fermi paradox by arguing that neither human nor extra-terrestrial civilizations can escape the time window singularity which, they claim, results from the way in which social characteristics of civilizations follow super-linear growth curves of cities. We question if data at the city level necessarily can lead to conclusions at the civilization level. More specifically, we suggest ways in which learnings from research, foresight, diversity and effective future government might act outside of their model to regulate super-linear growth curves of civilizations, and thus substantively increase the likelihood of civilizations progressing towards higher levels of the Kardashev scale. Moreover, we believe their claimed history of the collapse of terrestrial societies used to evidence their model is difficult to justify. Overall, we cast reasonable doubt on the ability of their proposed model to satisfactorily explain the Fermi paradox.
... However, in response to SARS-CoV-2, the scientific community achieved remarkable breakthroughs, progressing from preclinical trials to global vaccination within months (Polack et al., 2020;Mallory et al., 2022). This swift response was driven by novel platforms such as messenger RNA (mRNA) and viral vector technologies, which bypassed the need for live virus cultures, enabling faster production and adaptability to emerging viral strains (Verbeke et al., 2021). ...
The transformative advancements in vaccine development, particularly during the COVID-19 pandemic, have ushered in a new era of rapid innovation in vaccine technology. This study provides an overview of the latest trends in vaccine development, with a focus on India’s contributions alongside global progress. We examine the adoption of novel vaccine platforms, including mRNA, viral vector, protein subunit, and DNA vaccines, and analyze India’s role as a major vaccine producer. Our analysis also addresses emerging methodologies, regulatory developments, and the potential implications of these technologies for future public health crises. Finally, we consider the future challenges and opportunities in vaccine development, including the need for equitable access, scalable solutions, and the integration of new delivery technologies.
... When introduced into human tissue, the vaccine contains either self-replicating RNA or messenger RNA (mRNA), which both cause cells to express the SARS-CoV-2 spike protein. This teaches the body how to identify and destroy the corresponding pathogen [5][6][7]. Some other types of vaccines like the Sputnik V COVID-19 vaccine use an adenovirus shell containing DNA that encodes a SARS-CoV-2 protein. ...
Background: One of best ways to repel harmful viruses like corona is using of repulsive force between spinors which are existed within structures of cells and viruses. These spins could be induced within virus and cell structures by external magnetic fields which are emitted from currents of ions within blood cells. Motions of charges and ions within blood vessels produces magnetic fields. These fields force on spinors within cell and virus structures within alveolus and make them parallel. These spinning viruses interact with triplet spins of oxygen molecules within alveolus. According to the Pauli exclusion principle, parallel spinors repel each other and anti parallel spinors attract each other. Thus the triplet state of spinors of oxygen molecules could help cells to repel viruses with the same induced spin states. On the other hand, by using external waves, one can induce virtual viruses with opposite induced spins within alveolus and cancel effects of real viruses. We use of this property in controlling viral diseases. Purpose: Our aim is to 1. propose a mathematical model which use of repulsive force between parallel spinors and attractive force between anti parallel spinors within structures of oxygen molecules; alveolar cells and viruses to control COVID-19 disease. 2. We also introduce a mechanism mathematically to induce virtual viruses with opposite spinor states around real viruses within alveolus. These virtual viruses cancel effects of real viruses and form harmless bubbles. 3. We introduce a quantum mask to use of spinor interactions and repel viruses. Method: In this model, hemoglobin molecules and their irons, take special spins from heart waves, move along vessels and induce them to spinors within the structure of other cells like alveolar ones. These spinors select oxygen triplet molecules with opposite spins and repel parallel spin ones. Also, spinors which form RNAs and proteins of viruses could take parallel or opposite spins of any external magnetic field. These spinning viral structures are attracted by opposite spinors of alveolar cells and repel the oxygen molecules with parallel spins. This causes to a decrease in number of needed oxygen molecules within human's body. To control these viruses, we build a system which includes: 1. A vessel of water and oxygen molecules which be located near the face and is open. 2. A vessel of viruses which is located far from the face and is closed. 3. A heater-cooler system which connects two vessels. Results: During the respiration, alveolar air molecules go out, collide with vessels, attract oxygen and viral molecules with opposite spins and repel parallel ones. Consequently, viruses which could be attracted by alveolar cells go away from the face and build a mask against any similar spinning virus in another end of system. On the other hand, spinors around the viruses in second and closed vessel which couldn't be attracted by alveolar cells, form some bubbles. By heating and cooling the system these bubble shapes are induced in open vessel of system, make virtual viruses and bubbles which fly towards the face and alveolus. These virtual viruses attract real viruses and make spin-less bubbles which are harmless and go out of human's body. We formulate the model and obtain related currents. Conclusion: By using repulsive forces between triplet oxygen states and spinors within viral structures and induction of virtual viruses with opposite spins around real viruses; harmful effects of these viruses could be cancelled. Because; spinors of viruses are surrounded by anti parallel spinors of oxygen molecules and virtual viruses and harmless bubbles and pairs are formed. We have formulate the mechanism and obtained frequencies of waves which induce virtual viruses within alveolus.
... When introduced into human tissue, the vaccine contains either self-replicating RNA or messenger RNA (mRNA), which both cause cells to express the SARS-CoV-2 spike protein. This teaches the body how to identify and destroy the corresponding pathogen [5][6][7]. Some other types of vaccines like the Sputnik V COVID-19 vaccine use an adenovirus shell containing DNA that encodes a SARS-CoV-2 protein. ...
Background: One of best ways to repel harmful viruses like corona is using of repulsive force between spinors which are existed within structures of cells and viruses. These spins could be induced within virus and cell structures by external magnetic fields which are emitted from currents of ions within blood cells. Motions of charges and ions within blood vessels produces magnetic fields. These fields force on spinors within cell and virus structures within alveolus and make them parallel. These spinning viruses interact with triplet spins of oxygen molecules within alveolus. According to the Pauli exclusion principle, parallel spinors repel each other and anti parallel spinors attract each other. Thus the triplet state of spinors of oxygen molecules could help cells to repel viruses with the same induced spin states. On the other hand, by using external waves, one can induce virtual viruses with opposite induced spins within alveolus and cancel effects of real viruses. We use of this property in controlling viral diseases. Purpose: Our aim is to 1. propose a mathematical model which use of repulsive force between parallel spinors and attractive force between anti parallel spinors within structures of oxygen molecules; alveolar cells and viruses to control COVID-19 disease. 2. We also introduce a mechanism mathematically to induce virtual viruses with opposite spinor states around real viruses within alveolus. These virtual viruses cancel effects of real viruses and form harmless bubbles. 3. We introduce a quantum mask to use of spinor interactions and repel viruses. Method: In this model, hemoglobin molecules and their irons, take special spins from heart waves, move along vessels and induce them to spinors within the structure of other cells like alveolar ones. These spinors select oxygen triplet molecules with opposite spins and repel parallel spin ones. Also, spinors which form RNAs and proteins of viruses could take parallel or opposite spins of any external magnetic field. These spinning viral structures are attracted by opposite spinors of alveolar cells and repel the oxygen molecules with parallel spins.
... Human isolated dendritic cells were tolerant to mammalian mRNA when exposed to mRNA from different sources, while a strong inflammatory cytokine response was detected when mRNA from bacterial and necrotic mammalian cells were delivered. [62,63] In conclusion, it is certain that exRNA has an effect on the immune system, but the influence of different types of exRNA, the different sources of exRNA and whether there is a carrier is still unknown. ...
The RNA found in the circular system is known as extracellular RNA (exRNA). This kind of RNA has been found to play a biological role similar to that of a messenger. They can be used as indicators of disease status or the physiological health of an organism. A large number of RNA‐based biomaterials have been developed by simulating the biological function and structure of natural RNA molecules. The structural programmability of RNA‐based biomaterials provides the spur for scientists to pioneer new approaches in disease detection and prevention. Nevertheless, the link between exRNA function and the design of RNA‐based biomaterials has not been fully understood. Understanding the biological structure and function of exRNA will contribute to the clinical translation of this novel biotechnology. The present review discusses the research progress associated with exRNA and their derivatives to bridge the gap between natural exRNA and RNA‐based biomaterials.
... However, many models overlook mRNA secondary structure, reducing performance in RNA degradation prediction 35 . The advancements in bioinformatics have highlighted the potential of the abovementioned approaches in predicting mRNA degradation, a crucial factor in designing stabilized RNA therapeutics [36][37][38] . RNA degradation prediction is crucial for mRNA stability, therapeutic applications, gene expression, and viral RNA research and is impacted by RNA secondary structure, with specific motifs affecting rates with recent advances, sequence and structural data have been integrated to improve prediction accuracy, as in COVID-19 vaccine mRNA stability models. ...
In this study, we introduce StructmRNA, a new BERT-based model that was designed for the detailed analysis of mRNA sequences and structures. The success of DNABERT in understanding the intricate language of non-coding DNA with bidirectional encoder representations is extended to mRNA with StructmRNA. This new model uses a special dual-level masking technique that covers both sequence and structure, along with conditional masking. This enables StructmRNA to adeptly generate meaningful embeddings for mRNA sequences, even in the absence of explicit structural data, by capitalizing on the intricate sequence-structure correlations learned during extensive pre-training on vast datasets. Compared to well-known models like those in the Stanford OpenVaccine project, StructmRNA performs better in important tasks such as predicting RNA degradation. Thus, StructmRNA can inform better RNA-based treatments by predicting the secondary structures and biological functions of unseen mRNA sequences. The proficiency of this model is further confirmed by rigorous evaluations, revealing its unprecedented ability to generalize across various organisms and conditions, thereby marking a significant advance in the predictive analysis of mRNA for therapeutic design. With this work, we aim to set a new standard for mRNA analysis, contributing to the broader field of genomics and therapeutic development.
... Proper optimization of vaccine mRNA can reduce the dosage required for each injection leading to more efficient immunization programs [5]. The basic structure of mRNA consists of a protein-encoding open reading frame (ORF), 5 and 3 untranslated regions (UTRs), a 7-methylguanosine 5 cap structure, and a 3 poly(A) tail [6]. To enhance protein expression, various elements of the mRNA are optimized. ...
Messenger RNA (mRNA) vaccines represent a groundbreaking advancement in immunology and public health, particularly highlighted by their role in combating the COVID-19 pandemic. Optimizing mRNA-based antigen expression is a crucial focus in this emerging industry. We have developed a bioinformatics tool named AntigenBoost to address the challenge posed by destabilizing dipeptides that hinder ribosomal translation. AntigenBoost identifies these dipeptides within specific antigens and provides a range of potential amino acid substitution strategies using a two-dimensional scoring system. Through a combination of bioinformatics analysis and experimental validation, we significantly enhanced the in vitro expression of mRNA-derived Respiratory Syncytial Virus fusion glycoprotein and Influenza A Hemagglutinin antigen. Notably, a single amino acid substitution improved the immune response in mice, underscoring the effectiveness of AntigenBoost in mRNA vaccine design.
Background:
We evaluated safety and immunogenicity of the first mRNA vaccines against potentially pandemic avian H10N8 and H7N9 influenza viruses.
Methods:
Two randomized, placebo-controlled, double-blind, phase 1 clinical trials enrolled participants between December 2015 and August 2017 at single centers in Germany (H10N8) and USA (H7N9). Healthy adults (ages 18-64 years for H10N8 study; 18-49 years for H7N9 study) participated. Participants received vaccine or placebo in a 2-dose vaccination series 3 weeks apart. H10N8 intramuscular (IM) dose levels of 25, 50, 75, 100, and 400 µg and intradermal dose levels of 25 and 50 µg were evaluated. H7N9 IM 10-, 25-, and 50-µg dose levels were evaluated; 2-dose series 6 months apart was also evaluated. Primary endpoints were safety (adverse events) and tolerability. Secondary immunogenicity outcomes included humoral (hemagglutination inhibition [HAI], microneutralization [MN] assays) and cell-mediated responses (ELISPOT assay).
Results:
H10N8 and H7N9 mRNA IM vaccines demonstrated favorable safety and reactogenicity profiles. No vaccine-related serious adverse event was reported. For H10N8 (N = 201), 100-µg IM dose induced HAI titers ≥ 1:40 in 100% and MN titers ≥ 1:20 in 87.0% of participants. The 25-µg intradermal dose induced HAI titers > 1:40 in 64.7% of participants compared to 34.5% of participants receiving the IM dose. For H7N9 (N = 156), IM doses of 10, 25, and 50 µg achieved HAI titers ≥ 1:40 in 36.0%, 96.3%, and 89.7% of participants, respectively. MN titers ≥ 1:20 were achieved by 100% in the 10- and 25-µg groups and 96.6% in the 50-µg group. Seroconversion rates were 78.3% (HAI) and 87.0% (MN) for H10N8 (100 µg IM) and 96.3% (HAI) and 100% (MN) in H7N9 (50 µg). Significant cell-mediated responses were not detected in either study.
Conclusions:
The first mRNA vaccines against H10N8 and H7N9 influenza viruses were well tolerated and elicited robust humoral immune responses. ClinicalTrials.gov NCT03076385 and NCT03345043.
Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability. Keywords: nucleoside modification, mRNA vaccine, HIV-1, rhesus macaque, neutralizing antibody, ADCC
Abstract In 1975, Milstein and Köhler revolutionized the medical world with the development of the hybridoma technique to produce monoclonal antibodies. Since then, monoclonal antibodies have entered almost every branch of biomedical research. Antibodies are now used as frontline therapeutics in highly divergent indications, ranging from autoimmune disease over allergic asthma to cancer. Wider accessibility and implementation of antibody-based therapeutics is however hindered by manufacturing challenges and high development costs inherent to protein-based drugs. For these reasons, alternative ways are being pursued to produce and deliver antibodies more cost-effectively without hampering safety. Over the past decade, messenger RNA (mRNA) based drugs have emerged as a highly appealing new class of biologics that can be used to encode any protein of interest directly in vivo. Whereas current clinical efforts to use mRNA as a drug are mainly situated at the level of prophylactic and therapeutic vaccination, three recent preclinical studies have addressed the feasibility of using mRNA to encode therapeutic antibodies directly in vivo. Here, we highlight the potential of mRNA-based approaches to solve several of the issues associated with antibodies produced and delivered in protein format. Nonetheless, we also identify key hurdles that mRNA-based approaches still need to take to fulfill this potential and ultimately replace the current protein antibody format.
CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400–1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD⁺CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8–6.3) and 10.8 months (8.1–16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.
mRNA vaccines have the potential to tackle many unmet medical needs that are unable to be addressed with conventional vaccine technologies. A potent and well-tolerated delivery technology is integral to fully realizing the potential of mRNA vaccines. Pre-clinical and clinical studies have demonstrated that mRNA delivered intramuscularly (IM) with first-generation lipid nanoparticles (LNPs) generates robust immune responses. Despite progress made over the past several years, there remains significant opportunity for improvement, as the most advanced LNPs were designed for intravenous (IV) delivery of siRNA to the liver. Here, we screened a panel of proprietary biodegradable ionizable lipids for both expression and immunogenicity in a rodent model when administered IM. A subset of compounds was selected and further evaluated for tolerability, immunogenicity, and expression in rodents and non-human primates (NHPs). A lead formulation was identified that yielded a robust immune response with improved tolerability. More importantly for vaccines, increased innate immune stimulation driven by LNPs does not equate to increased immunogenicity, illustrating that mRNA vaccine tolerability can be improved without affecting potency.
Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8 ⁺ T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.
Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection³. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4–6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically ‘cold’ tumour microenvironment⁸. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone—a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma—generated circulating polyfunctional neoantigen-specific CD4⁺ and CD8⁺ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.
Statement of significance:
The delivery of messenger RNA (mRNA) to cells is promising to treat a variety of diseases. Therefore, the mRNA is typically packed in small lipid particles or polymer particles that help the mRNA to reach the cytoplasm of the cells. These particles should bind and carry the mRNA in the extracellular environment (e.g. blood, peritoneal fluid, ...), but should release the mRNA again in the intracellular environment. In this paper, we evaluated a method (Fluorescence Correlation Spectroscopy) that allows for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production.
The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold nanoparticles.
Here, we report a pathogenic role for type I IFN (IFN-I) signaling in macrophages, and not β cells in the islets, for the development of type 1 diabetes (T1D). Following lymphocytic choriomeningitis (LCMV) infection in the Rip-LCMV-GP T1D model, macrophages accumulated near islets and in close contact to islet-infiltrating GP-specific (autoimmune) CD8+ T cells. Depletion of macrophages with clodronate liposomes or genetic ablation of Ifnar in macrophages aborted T1D, despite proliferation of GP-specific (autoimmune) CD8+ T cells. Histopathologically, disrupted IFNα/β receptor (IFNAR) signaling in macrophages resulted in restriction of CD8+ T cells entering into the islets with significant lymphoid accumulation around the islet. Collectively, these results provide evidence that macrophages via IFN-I signaling, while not entering the islets, are directly involved in interacting, directing, or restricting trafficking of autoreactive-specific T cells into the islets as an important component in causing T1D.