SARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell response in humans.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

CD4+ T cells exhibit distinct transcriptional phenotypes in the lymph nodes and blood following mRNA vaccination in humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mRNA vaccination induce robust CD4+ T cell responses. Using single-cell transcriptomics, here, we evaluated CD4+ T cells specific for the SARS-CoV-2 spike protein in the blood and draining lymph nodes (dLNs) of individuals 3 months and 6 months after vaccination with the BNT162b2 mRNA vaccine. We analyzed 1,277 spike-specific CD4+ T cells, including 238 defined using Trex, a deep learning-based reverse epitope mapping method to predict antigen specificity. Human dLN spike-specific CD4+ follicular helper T (TFH) cells exhibited heterogeneous phenotypes, including germinal center CD4+ TFH cells and CD4+IL-10+ TFH cells. Analysis of an independent cohort of SARS-CoV-2-infected individuals 3 months and 6 months after infection found spike-specific CD4+ T cell profiles in blood that were distinct from those detected in blood 3 months and 6 months after BNT162b2 vaccination. Our findings provide an atlas of human spike-specific CD4+ T cell transcriptional phenotypes in the dLNs and blood following SARS-CoV-2 vaccination or infection.

A vaccine-induced public antibody protects against SARS-CoV-2 and emerging variants.

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Germinal centre-driven maturation of B cell response to mRNA vaccination.

Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.

The structural basis for control of eukaryotic protein kinases.

Eukaryotic protein kinases are key regulators of cell processes. Comparison of the structures of protein kinase domains, both alone and in complexes, allows generalizations to be made about the mechanisms that regulate protein kinase activation. Protein kinases in the active state adopt a catalytically competent conformation upon binding of both the ATP and peptide substrates that has led to an understanding of the catalytic mechanism. Docking sites remote from the catalytic site are a key feature of several substrate recognition complexes. Mechanisms for kinase activation through phosphorylation, additional domains or subunits, by scaffolding proteins and by kinase dimerization are discussed.

Testosterone Treatment and Fractures in Men with Hypogonadism.

BACKGROUND: Testosterone treatment in men with hypogonadism improves bone density and quality, but trials with a sufficiently large sample and a sufficiently long duration to determine the effect of testosterone on the incidence of fractures are needed. METHODS: In a subtrial of a double-blind, randomized, placebo-controlled trial that assessed the cardiovascular safety of testosterone treatment in middle-aged and older men with hypogonadism, we examined the risk of clinical fracture in a time-to-event analysis. Eligible men were 45 to 80 years of age with preexisting, or high risk of, cardiovascular disease; one or more symptoms of hypogonadism; and two morning testosterone concentrations of less than 300 ng per deciliter (10.4 nmol per liter), in fasting plasma samples obtained at least 48 hours apart. Participants were randomly assigned to apply a testosterone or placebo gel daily. At every visit, participants were asked if they had had a fracture since the previous visit. If they had, medical records were obtained and adjudicated. RESULTS: The full-analysis population included 5204 participants (2601 in the testosterone group and 2603 in the placebo group). After a median follow-up of 3.19 years, a clinical fracture had occurred in 91 participants (3.50%) in the testosterone group and 64 participants (2.46%) in the placebo group (hazard ratio, 1.43; 95% confidence interval, 1.04 to 1.97). The fracture incidence also appeared to be higher in the testosterone group for all other fracture end points. CONCLUSIONS: Among middle-aged and older men with hypogonadism, testosterone treatment did not result in a lower incidence of clinical fracture than placebo. The fracture incidence was numerically higher among men who received testosterone than among those who received placebo. (Funded by AbbVie and others; TRAVERSE ClinicalTrials.gov number, NCT03518034.).

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome.

Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. Here, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS), which enables Ψ-to-C transitions, for quantitative profiling of Ψ at single-base resolution. BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS detected all known Ψ sites in human rRNA and spliceosomal small nuclear RNAs and generated the quantitative Ψ map of human small nucleolar RNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine editing sites and N1-methyladenosine. Depletion of pseudouridine synthases TRUB1, PUS7 and PUS1 elucidated their targets and sequence motifs. We further identified a highly abundant Ψ114 site in Epstein-Barr virus-encoded small RNA EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation across virus families.

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans.

Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies1-7. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-28-10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11-13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses.

SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-191-5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene1. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity.

BNT162b vaccines protect rhesus macaques from SARS-CoV-2.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).

Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development.

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

The 10,000-year biocultural history of fallow deer and its implications for conservation policy.

Over the last 10,000 y, humans have manipulated fallow deer populations with varying outcomes. Persian fallow deer (Dama mesopotamica) are now endangered. European fallow deer (Dama dama) are globally widespread and are simultaneously considered wild, domestic, endangered, invasive and are even the national animal of Barbuda and Antigua. Despite their close association with people, there is no consensus regarding their natural ranges or the timing and circumstances of their human-mediated translocations and extirpations. Our mitochondrial analyses of modern and archaeological specimens revealed two distinct clades of European fallow deer present in Anatolia and the Balkans. Zooarchaeological evidence suggests these regions were their sole glacial refugia. By combining biomolecular analyses with archaeological and textual evidence, we chart the declining distribution of Persian fallow deer and demonstrate that humans repeatedly translocated European fallow deer, sourced from the most geographically distant populations. Deer taken to Neolithic Chios and Rhodes derived not from nearby Anatolia, but from the Balkans. Though fallow deer were translocated throughout the Mediterranean as part of their association with the Greco-Roman goddesses Artemis and Diana, deer taken to Roman Mallorca were not locally available Dama dama, but Dama mesopotamica. Romans also initially introduced fallow deer to Northern Europe but the species became extinct and was reintroduced in the medieval period, this time from Anatolia. European colonial powers then transported deer populations across the globe. The biocultural histories of fallow deer challenge preconceptions about the divisions between wild and domestic species and provide information that should underpin modern management strategies.

Understanding the Genetic Basis of C4 Kranz Anatomy with a View to Engineering C3 Crops.

One of the most remarkable examples of convergent evolution is the transition from C3 to C4 photosynthesis, an event that occurred on over 60 independent occasions. The evolution of C4 is particularly noteworthy because of the complexity of the developmental and metabolic changes that took place. In most cases, compartmentalized metabolic reactions were facilitated by the development of a distinct leaf anatomy known as Kranz. C4 Kranz anatomy differs from ancestral C3 anatomy with respect to vein spacing patterns across the leaf, cell-type specification around veins, and cell-specific organelle function. Here we review our current understanding of how Kranz anatomy evolved and how it develops, with a focus on studies that are dissecting the underlying genetic mechanisms. This research field has gained prominence in recent years because understanding the genetic regulation of Kranz may enable the C3-to-C4 transition to be engineered, an endeavor that would significantly enhance crop productivity.

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

Climate change challenges, plant science solutions.

Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an important role in developing crops with enhanced resilience to harsh conditions (e.g. heat, drought, salt stress, flooding, disease outbreaks) and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community.

Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily.

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3' and 5' flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5' ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.

Capturing the Onset of PRC2-Mediated Repressive Domain Formation.

Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.

Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.