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

Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

Monoclonal antibodies are increasingly used to prevent and treat viral infections and are pivotal in pandemic response efforts. Antibody-secreting cells (ASCs; plasma cells and plasmablasts) are an excellent source of high-affinity antibodies with therapeutic potential. Current methods to study antigen-specific ASCs either have low throughput, require expensive and labour-intensive screening or are technically demanding and therefore not widely accessible. Here, we present a straightforward technology for the rapid discovery of monoclonal antibodies from ASCs. Our approach combines microfluidic encapsulation of single cells into an antibody capture hydrogel with antigen bait sorting by conventional flow cytometry. With our technology, we screened millions of mouse and human ASCs and obtained anti-SARS-CoV-2 monoclonal antibodies with high affinity (85% of characterised antibodies bound the target). By facilitating access to the underexplored ASC compartment, the approach enables efficient antibody discovery as well as immunological studies into the generation of protective antibodies.This work was supported by the EU Horizon 2020 programme (ERC Advanced Investigator Awards 69566, to F.H. and EU H2020 Marie Skłodowska-Curie Individual Fellowship (MSCA-IF 750772) to T.S.K), the MRC (grant ref. MC_UU_00025/12, to J.E.D.T and TSF ref. MR/T032413/1, to N.J.M, and a scholarship to K.F.), the Medical Research Foundation (MRF-057-0002-RG-THAV-C0798, to J.E.D.T.), the NIHR Cambridge BRC (to J.E.D.T.), NHSBT (grant ref. WPA15-02, to N.J.M), the Wellcome Trust (ISSF ref. 204845/Z/16/Z, to N.J.M), Addenbrooke’s Charitable Trust (grant ref. 900239, to N.J.M), a scholarship from the Cambridge European Trust (to K.F.), a scholarship from St. John’s College Cambridge (to K.F.), a Postdoctoral Research fellowship by Boehringer Ingelheim (to M.I.J.R), an AstraZeneca studentship (to T.N.K), and a Gates Cambridge Scholarship (to G.L.P)

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