The mTOR protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR-dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of PI3K typical of cells lacking TSC2, a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics which may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days, and clearance of 0.22 mL/hr/kg, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study Day 6 following viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

The emerging new VOC B.1.1.529 (Omicron) variant has raised serious concerns due to multiple mutations, reported significant immune escape, and unprecedented rapid spreading speed. Currently, studies describing the neutralization ability of different homologous and heterologous booster vaccination against Omicron are still lacking. In this study, we explored the immunogenicity of COVID-19 breakthrough patients, BBIBP-CorV homologous booster group and BBIBP-CorV/ZF2001 heterologous booster group against SARS-CoV-2 pseudotypes corresponding to the prototype, Beta, Delta, and the emergent Omicron variant. Notably, at 14 days post two-dose inactivated vaccines, pVNT titre increased to 67.4 GMTs against prototype, 8.85 against Beta and 35.07 against Delta, while neutralization activity against Omicron was below the lower limit of quantitation in 80% of the samples. At day 14 post BBIBP-CorV homologous booster vaccination, GMTs of pVNT significantly increased to 285.6, 215.7, 250.8, 48.73 against prototype, Beta, Delta, and Omicron, while at day 14 post ZF2001 heterologous booster vaccination, GMTs of pVNT significantly increased to 1436.00, 789.6, 1501.00, 95.86, respectively. Post booster vaccination, 100% samples showed positive neutralization activity against Omicron, albeit illustrated a significant reduction (5.86- to 14.98-fold) of pVNT against Omicron compared to prototype at 14 days after the homologous or heterologous vaccine boosters. Overall, our study demonstrates that vaccine-induced immune protection might more likely be escaped by Omicron compared to prototypes and other VOCs. After two doses of inactivated whole-virion vaccines as the “priming” shot, a third heterologous protein subunit vaccine and a homologous inactivated vaccine booster could improve neutralization against Omicron.

The first tumor-specific shared antigens and the cancer-germline genes that code for these antigens were identified with antitumor cytolytic T lymphocytes obtained from cancer patients. A few HLA class I-restricted antigenic peptides were identified by this 'direct approach'. A large set of additional cancer-germline genes have now been identified by purely genetic approaches or by screening tumor cDNA expression libraries with the serum of cancer patients. As a result, a vast number of sequences are known that can code for tumor-specific shared antigens, but most of the encoded antigenic peptides have not yet been identified. We review here recent 'reverse immunology' approaches for the identification of new antigenic peptides. They are based on in vitro stimulation of naive T cells with dendritic cells that have either been loaded with a cancer-germline protein or that have been transduced with viruses carrying cancer-germline coding sequences. These approaches have led to the identification of many new antigenic peptides presented by class I or class II molecules. We also describe some aspects of the processing and presentation of these antigenic peptides.

Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans. IMPORTANCE Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle formation confirmed structural integrity. Group 2 HA stem antigen designs were identified that, when displayed on ferritin nanoparticles, activated B cells expressing inferred unmutated common ancestor (UCA) versions of human antibody lineages associated with cross-group-reactive, broadly neutralizing antibodies (bNAbs). Immunization of mice led to protection against a lethal homosubtypic influenza virus challenge. These candidate vaccines are now being manufactured for clinical evaluation.

Long noncoding RNAs (lncRNAs) mediate important epigenetic regulation in a wide range of biological processes and diseases. We applied comprehensive analyses of RNA-seq and CAGE-seq (cap analysis of gene expression and sequencing) to characterize the dynamic changes in lncRNA expression in rhesus macaque (Macaca mulatta) brain in four representative age groups. We identified 18 anatomically diverse lncRNA modules and 14 mRNA modules representing spatial, age, and sex specificities. Spatiotemporal-and sex-biased changes in lncRNA expression were generally higher than those observed in mRNA expression. A negative correlation between lncRNA and mRNA expression in cerebral cortex was observed and functionally validated. Our findings offer a fresh insight into spatial-, age-, and sex-biased changes in lncRNA expression in macaque brain and suggest that the changes represent a previously unappreciated regulatory system that potentially contributes to brain development and aging.

With B.1.1.529 SARS-CoV-2 variant's rapid spread and substantially increased resistance to neutralization by vaccinee and convalescent sera, monoclonal antibodies with potent neutralization are eagerly sought. To provide insight into effective neutralization, we determined cryo-EM structures and evaluated potent receptor-binding domain (RBD) antibodies for their ability to bind and neutralize this new variant. B.1.1.529 RBD mutations altered 16% of the RBD surface, clustering on a ridge of this domain proximal to the ACE2-binding surface and reducing binding of most antibodies. Significant inhibitory activity was retained, however, by select monoclonal antibodies including A19-58.1, B1-182.1, COV2-2196, S2E12, A19-46.1, S309 and LY-CoV1404, which accommodated these changes and neutralized B.1.1.529 with IC50s between 5.1-281 ng/ml, and we identified combinations of antibodies with potent synergistic neutralization. Structure-function analyses delineated the impact of resistance mutations and revealed structural mechanisms for maintenance of potent neutralization against emerging variants.

Graphical AbstractHighlights d The epitope for the neutralizing antibody G2 is confined to the apex of the MERS-CoV S1-NTD d G2 epitope is relatively well conserved d G2 IgG and Fab both neutralize pseudotyped and authentic MERS-CoV d G2 neutralizes by preventing the binding of DPP4 to trimeric S protein In Brief Wang et al. report the structural and functional characterization of the Middle East respiratory syndrome coronavirus (MERS-CoV)-neutralizing antibody G2.G2 recognizes a conserved epitope on the MERS-CoV S1 N-terminal domain (S1-NTD) and neutralizes MERS-CoV by interfering with binding to host receptor dipeptidyl peptidase-4 (DPP4). The findings are relevant for understanding the viral attachment mechanism and for the development of S1-NTD-based vaccines. SUMMARYMiddle East respiratory syndrome coronavirus (MERS-CoV) emerged into the human population in 2012 and has caused substantial morbidity and mortality. Potently neutralizing antibodies targeting the receptor-binding domain (RBD) on MERS-CoV spike (S) protein have been characterized, but much less is known about antibodies targeting non-RBD epitopes. Here, we report the structural and functional characterization of G2, a neutralizing antibody targeting the MERS-CoV S1 N-terminal domain (S1-NTD). Structures of G2 alone and in complex with the MERS-CoV S1-NTD define a site of vulnerability comprising two loops, each of which contain a residue mutated in G2-escape variants. Cell-surface binding studies and in vitro competition experiments demonstrate that G2 strongly disrupts the attachment of MERS-CoV S to its receptor, dipeptidyl peptidase-4 (DPP4), with the inhibition requiring the native trimeric S conformation. These results advance our understanding of antibody-mediated neutralization of coronaviruses and should facilitate the development of immunotherapeutics and vaccines against MERS-CoV.