SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response
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ABSTRACT:
PROVIDER: PRJEB53190 | ENA |
REPOSITORIES: ENA
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Project description:Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Moderna’s mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (mΨ), is terminated by a long poly(A) tail (~100 nucleotides) followed by an mΨCmΨAG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of mΨCmΨAG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after mΨCmΨAG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after mΨCmΨAG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.
2025-01-09 | GSE233059 | GEO
Project description:SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response
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Project description:Chimeric Antigen Receptor (CAR) T-cell therapy effectively treats human cancer, but loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T-cells triggers engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR-T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited priming of endogenous anti-tumor T-cells (antigen spreading). This process was accompanied by shifts in CAR-T metabolism toward oxidative phosphorylation and was critically dependent on CAR T-derived IFN-γ. Antigen spreading induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR-antigen-negative, and heterogenous tumor control was further enhanced by genetically amplifying CAR T IFN-γ expression. Thus, CAR T-cell-derived IFN-γ plays a critical role in promoting antigen spreading, and vaccine boosting provides a clinically-translatable strategy to drive such responses against solid tumors.
2023-05-17 | GSE212453 | GEO
Project description:The development of effective mRNA vaccines for breast cancer has been constrained by the lack of highly immunogenic and tumor-specific antigens. Here, we identify PDE6G, an immune-privileged retina-enriched antigen aberrantly overexpressed in breast tumors, and develop a PDE6G mRNA-LNP vaccine. Vaccination elicited robust CD8+ T cell responses, promoted clonal expansion of effector and IFN-competent T cell subsets, and suppressed tumor growth without detectable toxicity. Single-cell RNA sequencing (scRNA-seq) revealed that vaccination of PDE6G mRNA-LNP reshapes the tumor immune microenvironment (TIME) by expanding antigen-presenting Cd74+ tumor-associated macrophages (TAMs) while reducing immunosuppressive Maf+ macrophages, and enhancing crosstalk between macrophages and CD8+ T cells. Moreover, combining PDE6G mRNA-LNP with PD-1 blockade further improved anti-tumor efficacy. These findings establish cancer-retina antigens as promising vaccine targets and demonstrate that PDE6G mRNA-LNP coordinates adaptive immunity and tumor microenvironment remodeling, providing a safe and potent strategy for breast cancer immunotherapy.
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Project description:Immunotherapy has demonstrated durable responses in a subset of patients with head and neck cancer, offering potential survival benefits to those with poor prognoses and resistance to first-line treatments. Despite this, only a modest 15-20% are shown to respond. Cancer vaccines have recently demonstrated efficacy in some solid tumours including melanoma, as well as immune-cold pancreatic and liver cancer, inducing a cytotoxic T cell response and eradicating cancer cells. Our previous research identified cancer testis antigens MAGED4B and FJX1 as highly immunogenic and overexpressed in the majority of HPV-independent head-and-neck squamous cell carcinoma (HPVnegHNSCC) patients. In this study, we developed a novel DNA vaccine utilising the non-plasmid doggyboneTM (dbDNATM) platform to deliver these antigens for targeting head and neck cancer. A series of doggyboneTM vaccine prototypes targeting MAGED4B and FJX1 were generated both to optimise the backbone and to present antigens in an immunogenic form. Vaccine immunogenicity and cytotoxicity were accessed using interferon-gamma. Enzyme-Linked Immunospot (ELISpot) assay. Vaccine efficacy was evaluated in the MOC-2 oral cancer model through tumour monitoring, assessing the infiltration of CD4+, CD8+ and regulatory T cells by immunohistochemistry as well as RNAseq analysis. The optimised dual antigen dbDNA DNA vaccine significantly reduced tumour size. Furthermore, combining the vaccine with anti-PD1 therapy significantly delayed tumour growth and metastasis, thereby enhancing survival in tumour-bearing mice. Vaccination resulted in significant recruitment of CD4+ and CD8+ T cells but not regulatory T cells. IHC analysis also confirmed the ability of dbDNA™ vaccine TGL-100 OPT in shifting M2 to M1 polarisation, highlighting their therapeutic promise. Transcriptomic analysis of immune cells in the tumour indicates that vaccination promotes long term anti-tumour responses by upregulating memory T cell genes T cell factor-7 (Tcf7) and lymphoid enhancer binding factor 1 (Lef1) while suppressing PDL-1 and inhibiting anti-inflammatory cytokines such as IL-10 and IL-27. These findings suggest that our cancer vaccine effectively recruits cytotoxic T cells to immune cold tumours, minimising the host’s immune inhibitory mechanisms within the tumour and resulting in long-term protection against head and neck cancer.
2025-12-01 | GSE310571 | GEO
Project description:Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of diarrheal illness in low- and middle-income countries. The live-attenuated ACE527 vaccine, adjuvanted with double mutant LT (dmLT), affords clear but partial protection against ETEC challenge inhuman volunteers. Comparatively, initial wild-type ETEC challenge completely protects against severe diarrhea on homologous re-challenge...To investigate molecular determinants of protection, vaccine antigen content was compared to wild-type ETEC, and proteome microarrays were used to assess immune responses following vaccination and ETEC challenge... Although molecular interrogation of the vaccine confirmed expression of targeted canonical antigens, relative to wild-type ETEC, vaccine strains were deficient in production of flagellar antigens, immotile, and lacked production of the EtpA adhesin. Similarly, vaccination ± dmLT elicited responses to targeted canonical antigens, but relative to wild-type challenge, vaccine responses to some potentially protective non-canonical antigens including EtpA were diminished or absent...These studies highlight important differences in vaccine and wild-type ETEC antigen content and call attention to distinct immunologic signatures that could inform investigation of correlates of protection, and guide vaccine antigen selection for these pathogens of global importance.
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Project description:Given the importance of sustained antigen presentation in maintenance of lymph node (LN) immune responses, we hypothesized that vaccine antigen availability and antigen-presenting cell (APC) populations may affect LN expansion. Compared to LNs of mice given the full MPS vaccine, LNs of mice given an MPS vaccine without antigen became prominently less enlarged and contracted sooner.To identify potential mediators of this differential, antigen-dependent response, we next focused the analysis of our scRNA-seq dataset on LN APC populations.
2024-04-05 | E-MTAB-13698 | biostudies-arrayexpress
Project description:Waning immunity and the emergence of immune evasive SARS-CoV-2 variants jeopardize vaccine efficacy and contribute to breakthrough infections. The immune response that promotes vaccine-induced protection against SARS-CoV-2 breakthrough infection remains poorly understood. To model breakthrough infections, we vaccinated mice with a low dose vaccine formulation containing MF59-like adjuvant with prefusion-stabilized SARS-CoV-2 spike protein and challenged with immune evasive B.1.351 variant. Here, we show that eosinophils are required for protection against SARS-CoV-2 during a vaccine breakthrough infection. We found that vaccine breakthrough infection leads to a 2-log reduction in lung viral burden with restricted replication within the large airways as compared to naïve infected mice. Despite reduced antiviral gene expression, infected vaccinated mice show increased immune cell infiltration, characterized by monocytes, interstitial macrophages, and eosinophils, but with reduced activation markers into the lung parenchyma as compared to infected naïve mice. Single cell RNA-seq revealed that viral RNA was highly associated with eosinophils that corresponded to an IFN-γ biased phenotype and expression of antiviral genes including Cystatin B, an inhibitor of cysteine protease involved in SARS-CoV-2 entry via the endosome. Monocytes from infected vaccinated, but not naïve, mice showed high expression for eosinophil chemoattractant Ccl24 (eotaxin-2). Antibody-mediated depletion of eosinophils prior to infection of vaccinated mice resulted in increased virus replication and viral antigen staining deep in the lungs as compared to isotype control infected vaccinated mice. These results demonstrate the importance of eosinophils in vaccine-mediated protection and highlight the need for durable antibody responses that protect against lung infection and inflammation.
2025-08-08 | GSE274118 | GEO
Project description:<p><em>Bordetella pertussis</em> is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of <em>B. pertussis</em> physiology during <em>in vitro</em> cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of <em>B. pertussis</em>. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of <em>B. pertussis</em> (BvgASR) was not evidenced as the sole virulence regulator in this <em>in vitro</em> growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to <em>B. pertussis</em> culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.</p>
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