N 1 -methylpseudouridylation of mRNA causes +1 ribosomal frameshifting

In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1, 2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3–5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization

Age-associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade

Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination

N 1 -methylpseudouridylation of mRNA causes +1 ribosomal frameshifting

Acknowledgements: A.E.W. and T.P. are supported by the Medical Research Council, grant number MC_UU_00025/7(A.E.W.). J.C.Y.-P., E.H., A.P.F. and J.E.D.T. are supported by the Medical Research Council (RG95376 and MC_UU_00025/12). T.E.M. was financially supported by the Integrative Toxicology Training Partnership. T.E.M., M.R., T.V.d.H., C.M.S., J.E.D.T., K.S.L. and A.E.W. acknowledge funding from Wellcome Leap as part of the R3 Program. PITCH was funded by the UK Department of Health and Social Care and UKRI (MR/W02067X/1 and MR/X009297/1), with contributions from UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Huo Family Foundation and The National Institute for Health Research (COV19-RECPLAS). In Liverpool PITCH is a sub-study of UKHSA’s SIREN study. P.K. is an NIHR Senior Investigators and is funded by WT109965MA. S.J.D. is funded by an NIHR Global Research Professorship (NIHR300791). L.T. is supported by the Wellcome Trust (grant number 205228/Z/16/Z), the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections (EZI) (NIHR200907) and the Centre of Excellence in Infectious Diseases Research (CEIDR) and the Alder Hey Charity. This research was supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. The authors thank the MRC Toxicology Unit Proteomics Facility for assistance with mass spectrometry analysis and A. Chong and D. Launer for assistance with DNA extraction and HLA typing.In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1, 2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3–5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization

N 1 -methylpseudouridylation of mRNA causes +1 ribosomal frameshifting

Acknowledgements: A.E.W. and T.P. are supported by the Medical Research Council, grant number MC_UU_00025/7(A.E.W.). J.C.Y.-P., E.H., A.P.F. and J.E.D.T. are supported by the Medical Research Council (RG95376 and MC_UU_00025/12). T.E.M. was financially supported by the Integrative Toxicology Training Partnership. T.E.M., M.R., T.V.d.H., C.M.S., J.E.D.T., K.S.L. and A.E.W. acknowledge funding from Wellcome Leap as part of the R3 Program. PITCH was funded by the UK Department of Health and Social Care and UKRI (MR/W02067X/1 and MR/X009297/1), with contributions from UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Huo Family Foundation and The National Institute for Health Research (COV19-RECPLAS). In Liverpool PITCH is a sub-study of UKHSA’s SIREN study. P.K. is an NIHR Senior Investigators and is funded by WT109965MA. S.J.D. is funded by an NIHR Global Research Professorship (NIHR300791). L.T. is supported by the Wellcome Trust (grant number 205228/Z/16/Z), the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections (EZI) (NIHR200907) and the Centre of Excellence in Infectious Diseases Research (CEIDR) and the Alder Hey Charity. This research was supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. The authors thank the MRC Toxicology Unit Proteomics Facility for assistance with mass spectrometry analysis and A. Chong and D. Launer for assistance with DNA extraction and HLA typing.In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1, 2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3–5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization

N 1 -methylpseudouridylation of mRNA causes +1 ribosomal frameshifting

Acknowledgements: A.E.W. and T.P. are supported by the Medical Research Council, grant number MC_UU_00025/7(A.E.W.). J.C.Y.-P., E.H., A.P.F. and J.E.D.T. are supported by the Medical Research Council (RG95376 and MC_UU_00025/12). T.E.M. was financially supported by the Integrative Toxicology Training Partnership. T.E.M., M.R., T.V.d.H., C.M.S., J.E.D.T., K.S.L. and A.E.W. acknowledge funding from Wellcome Leap as part of the R3 Program. PITCH was funded by the UK Department of Health and Social Care and UKRI (MR/W02067X/1 and MR/X009297/1), with contributions from UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Huo Family Foundation and The National Institute for Health Research (COV19-RECPLAS). In Liverpool PITCH is a sub-study of UKHSA’s SIREN study. P.K. is an NIHR Senior Investigators and is funded by WT109965MA. S.J.D. is funded by an NIHR Global Research Professorship (NIHR300791). L.T. is supported by the Wellcome Trust (grant number 205228/Z/16/Z), the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections (EZI) (NIHR200907) and the Centre of Excellence in Infectious Diseases Research (CEIDR) and the Alder Hey Charity. This research was supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. The authors thank the MRC Toxicology Unit Proteomics Facility for assistance with mass spectrometry analysis and A. Chong and D. Launer for assistance with DNA extraction and HLA typing.In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1, 2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3–5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization

N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.

Acknowledgements: A.E.W. and T.P. are supported by the Medical Research Council, grant number MC_UU_00025/7(A.E.W.). J.C.Y.-P., E.H., A.P.F. and J.E.D.T. are supported by the Medical Research Council (RG95376 and MC_UU_00025/12). T.E.M. was financially supported by the Integrative Toxicology Training Partnership. T.E.M., M.R., T.V.d.H., C.M.S., J.E.D.T., K.S.L. and A.E.W. acknowledge funding from Wellcome Leap as part of the R3 Program. PITCH was funded by the UK Department of Health and Social Care and UKRI (MR/W02067X/1 and MR/X009297/1), with contributions from UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Huo Family Foundation and The National Institute for Health Research (COV19-RECPLAS). In Liverpool PITCH is a sub-study of UKHSA’s SIREN study. P.K. is an NIHR Senior Investigators and is funded by WT109965MA. S.J.D. is funded by an NIHR Global Research Professorship (NIHR300791). L.T. is supported by the Wellcome Trust (grant number 205228/Z/16/Z), the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections (EZI) (NIHR200907) and the Centre of Excellence in Infectious Diseases Research (CEIDR) and the Alder Hey Charity. This research was supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. The authors thank the MRC Toxicology Unit Proteomics Facility for assistance with mass spectrometry analysis and A. Chong and D. Launer for assistance with DNA extraction and HLA typing.In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization

N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.

In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization

Recycling of memory B cells between germinal center and lymph node subcapsular sinus supports affinity maturation to antigenic drift.

Infection or vaccination leads to the development of germinal centers (GC) where B cells evolve high affinity antigen receptors, eventually producing antibody-forming plasma cells or memory B cells. Here we follow the migratory pathways of B cells emerging from germinal centers (BEM) and find that many BEM cells migrate into the lymph node subcapsular sinus (SCS) guided by sphingosine-1-phosphate (S1P). From the SCS, BEM cells may exit the lymph node to enter distant tissues, while some BEM cells interact with and take up antigen from SCS macrophages, followed by CCL21-guided return towards the GC. Disruption of local CCL21 gradients inhibits the recycling of BEM cells and results in less efficient adaption to antigenic variation. Our findings thus suggest that the recycling of antigen variant-specific BEM cells and transport of antigen back to GC may support affinity maturation to antigenic drift

Stability of gut microbiome after COVID-19 vaccination in healthy and immuno-compromised individuals.

Bidirectional interactions between the immune system and the gut microbiota are key contributors to various physiological functions. Immune-associated diseases such as cancer and autoimmunity, and efficacy of immunomodulatory therapies, have been linked to microbiome variation. Although COVID-19 infection has been shown to cause microbial dysbiosis, it remains understudied whether the inflammatory response associated with vaccination also impacts the microbiota. Here, we investigate the temporal impact of COVID-19 vaccination on the gut microbiome in healthy and immuno-compromised individuals; the latter included patients with primary immunodeficiency and cancer patients on immunomodulating therapies. We find that the gut microbiome remained remarkably stable post-vaccination irrespective of diverse immune status, vaccine response, and microbial composition spanned by the cohort. The stability is evident at all evaluated levels including diversity, phylum, species, and functional capacity. Our results indicate the resilience of the gut microbiome to host immune changes triggered by COVID-19 vaccination and suggest minimal, if any, impact on microbiome-mediated processes. These findings encourage vaccine acceptance, particularly when contrasted with the significant microbiome shifts observed during COVID-19 infection

Age-associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade

Acknowledgements: This work was funded by the UK Medical Research Council (project number MC_UU_00025/12), the Medical Research Foundation (MRF-057-0002-RG-THAV-C0798) and The Evelyn Trust (grant number 20/40) to J.E.D.T. N.J.M. was supported by the MRC (TSF ref. MR/T032413/1), NHSBT (grant ref. WPA15-02) and Addenbrooke’s Charitable Trust (grant ref. 900239). M.A.C. was supported by the Medical Research Council (project number MC_UU_00025/10). K.R.P. was supported by the Medical Research Council (project number MC_UU_00025/11). K.F. held an MRC studentship with support from the Cambridge European Trust and St. John’s College. K.W. has received funding by the Deutsche Forschungsgemeinschaft (WA 1597/6-1 and WA 1597/7-1). K.W. and B.K. received support by the German Federal Ministry of Education and Research (BMBF) through a grant to the German genetic multi-organ Auto-Immunity Network (GAIN), grant code 01GM2206A. F.H. is an ERC Advanced Investigator (695669). We thank Carola G. Vinuesa for helpful discussion. The authors also thank the Flow Cytometry Facilities at the MRC-Toxicology Unit, University of Cambridge; Katarzyna Kania from CRUK-CI-Genomics, Cambridge UK for advice on single cell RNA sequencing experiments; and Rosalind Kieran from the Department of Oncology, Cambridge University NHS Hospitals Foundation Trust, Cambridge UK, for contributions for patient recruitment and data collection.Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination