Reducing cell intrinsic immunity to mRNA vaccine alters adaptive immune responses in mice.

The response to mRNA vaccines needs to be sufficient for immune cell activation and recruitment, but moderate enough to ensure efficacious antigen expression. The choice of the cap structure and use of N1-methylpseudouridine (m1Ψ) instead of uridine, which have been shown to reduce RNA sensing by the cellular innate immune system, has led to improved efficacy of mRNA vaccine platforms. Understanding how RNA modifications influence the cell intrinsic immune response may help in the development of more effective mRNA vaccines. In the current study, we compared mRNA vaccines in mice against influenza virus using three different mRNA formats: uridine-containing mRNA (D1-uRNA), m1Ψ-modified mRNA (D1-modRNA), and D1-modRNA with a cap1 structure (cC1-modRNA). D1-uRNA vaccine induced a significantly different gene expression profile to the modified mRNA vaccines, with an up-regulation of Stat1 and RnaseL, and increased systemic inflammation. This result correlated with significantly reduced antigen-specific antibody responses and reduced protection against influenza virus infection compared with D1-modRNA and cC1-modRNA. Incorporation of m1Ψ alone without cap1 improved antibodies, but both modifications were required for the optimum response. Therefore, the incorporation of m1Ψ and cap1 alters protective immunity from mRNA vaccines by altering the innate immune response to the vaccine material.

ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model.

Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.

Progress in vaccine development for infectious diseases-a Keystone Symposia report.

The COVID-19 pandemic has taught us many things, among the most important of which is that vaccines are one of the cornerstones of public health that help make modern longevity possible. While several different vaccines have been successful at stemming the morbidity and mortality associated with various infectious diseases, many pathogens/diseases remain recalcitrant to the development of effective vaccination. Recent advances in vaccine technology, immunology, structural biology, and other fields may yet yield insight that will address these diseases; they may also help improve societies' preparedness for future pandemics. On June 1-4, 2022, experts in vaccinology from academia, industry, and government convened for the Keystone symposium "Progress in Vaccine Development for Infectious Diseases" to discuss state-of-the-art technologies, recent advancements in understanding vaccine-mediated immunity, and new aspects of antigen design to aid vaccine effectiveness.

Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice.

As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)-encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8+ T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors.

BACKGROUND: Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma for which targeted immunotherapies have been developed. This work aimed to characterize GPC2 expression in pediatric brain tumors and develop an mRNA CAR T cell approach against this target. METHODS: We investigated GPC2 expression across a cohort of primary pediatric brain tumor samples and cell lines using RNA sequencing, immunohistochemistry, and flow cytometry. To target GPC2 in the brain with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we used optimized mRNA to create transient chimeric antigen receptor (CAR) T cells. We developed four mRNA CAR T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment for preclinical testing. RESULTS: We identified high GPC2 expression across multiple pediatric brain tumor types including medulloblastomas, embryonal tumors with multilayered rosettes, other central nervous system embryonal tumors, as well as definable subsets of highly malignant gliomas. We next validated and prioritized CAR configurations using in vitro cytotoxicity assays with GPC2-expressing neuroblastoma cells, where the light-to-heavy single chain variable fragment configurations proved to be superior. We expanded the testing of the two most potent GPC2-directed CAR constructs to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, biweekly locoregional delivery of 2-4 million GPC2-directed mRNA CAR T cells induced significant tumor regression in an orthotopic medulloblastoma model and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma xenograft model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. CONCLUSION: Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells, laying the framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors.

A systematic dissection of determinants and consequences of snoRNA-guided pseudouridylation of human mRNA.

RNA can be extensively modified post-transcriptionally with >170 covalent modifications, expanding its functional and structural repertoire. Pseudouridine (Ψ), the most abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA molecules. It remains unclear whether pseudouridylation of mRNA can be snoRNA-guided, bearing important implications for understanding the physiological target spectrum of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here, using a massively parallel reporter based strategy we simultaneously interrogate Ψ levels across hundreds of synthetic constructs with predesigned complementarity against endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation can occur on mRNA targets. However, this is typically achieved at relatively low efficiencies, and is constrained by mRNA localization, snoRNA expression levels and the length of the snoRNA:mRNA complementarity stretches. We exploited these insights for the design of snoRNAs targeting pseudouridylation at premature termination codons, which was previously shown to suppress translational termination. However, in this and follow-up experiments in human cells we observe no evidence for significant levels of readthrough of pseudouridylated stop codons. Our study enhances our understanding of the scope, 'design rules', constraints and consequences of snoRNA-mediated pseudouridylation.

Developing mRNA for Therapy.

Messenger RNA was discovered in 1961 and it took 60 years until the first mRNA became FDA-approved product in the form of COVID-19 mRNA vaccine. During those years a lot of progress has been made by hundreds of scientists. It was 1978 when the first-time isolated mRNA delivered into mammalian cells produced the encoded protein. In vitro transcription introduced in 1984 made it possible to generate any desired mRNA from the encoding plasmid using phage RNA polymerases. In the early 90s mRNA was used for therapy as well as vaccine against infectious diseases and cancer. Inflammatory nature of the mRNAs limited their in vivo use. Replacing uridine with pseudouridine made the mRNA non-immunogenic, more stable and highly translatable. Delivery of the lipid nanoparticle-formulated nucleoside-modified mRNA encoding viral antigens became a platform for effective vaccine. Labile nature of the mRNA is ideal for transient production of the viral antigen, to generate effective antibody and cellular immune response. The mRNA platform is revolutionizing the delivery of effective and safe vaccines, therapeutics and gene therapies.

Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine.

Lipid nanoparticle (LNP)-formulated nucleoside-modified mRNA vaccines have proven to be very successful in the fight against the coronavirus disease 2019 (COVID-19) pandemic. They are effective, safe, and can be produced in large quantities. However, the long-term storage of mRNA-LNP vaccines without freezing is still a challenge. Here, we demonstrate that nucleoside-modified mRNA-LNPs can be lyophilized, and the physicochemical properties of the lyophilized material do not significantly change for 12 weeks after storage at room temperature and for at least 24 weeks after storage at 4°C. Importantly, we show in comparative mouse studies that lyophilized firefly luciferase-encoding mRNA-LNPs maintain their high expression, and no decrease in the immunogenicity of a lyophilized influenza virus hemagglutinin-encoding mRNA-LNP vaccine was observed after 12 weeks of storage at room temperature or for at least 24 weeks after storage at 4°C. Our studies offer a potential solution to overcome the long-term storage-related limitations of nucleoside-modified mRNA-LNP vaccines.

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA.

The presence of the cap structure on the 5'-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5'-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC-MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5' untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models.

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.

Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema.

Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.

BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans.

BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.

A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis.

The ability to control autoreactive T cells without inducing systemic immune suppression is the major goal for treatment of autoimmune diseases. The key challenge is the safe and efficient delivery of pharmaceutically well-defined antigens in a noninflammatory context. Here, we show that systemic delivery of nanoparticle-formulated 1 methylpseudouridine-modified messenger RNA (m1Ψ mRNA) coding for disease-related autoantigens results in antigen presentation on splenic CD11c+ antigen-presenting cells in the absence of costimulatory signals. In several mouse models of multiple sclerosis, the disease is suppressed by treatment with such m1Ψ mRNA. The treatment effect is associated with a reduction of effector T cells and the development of regulatory T cell (Treg cell) populations. Notably, these Treg cells execute strong bystander immunosuppression and thus improve disease induced by cognate and noncognate autoantigens.

COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses.

An effective vaccine is needed to halt the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase I/II coronavirus disease 2019 (COVID-19) vaccine trial with BNT162b1, a lipid nanoparticle-formulated nucleoside-modified mRNA that encodes the receptor binding domain (RBD) of the SARS-CoV-2 spike protein1. Here we present antibody and T cell responses after vaccination with BNT162b1 from a second, non-randomized open-label phase I/II trial in healthy adults, 18-55 years of age. Two doses of 1-50 µg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those seen in serum from a cohort of individuals who had recovered from COVID-19. Geometric mean titres of SARS-CoV-2 serum-neutralizing antibodies on day 43 were 0.7-fold (1-µg dose) to 3.5-fold (50-µg dose) those of the recovered individuals. Immune sera broadly neutralized pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had T helper type 1 (TH1)-skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon-γ was produced by a large fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest that it has the potential to protect against COVID-19 through multiple beneficial mechanisms.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.