Influenza virus can escape most antibodies with single mutations. However, rare antibodies broadly neutralize many viral strains. It is unclear how easily influenza virus might escape such antibodies ...if there was strong pressure to do so. Here, we map all single amino-acid mutations that increase resistance to broad antibodies to H1 hemagglutinin. Our approach not only identifies antigenic mutations but also quantifies their effect sizes. All antibodies select mutations, but the effect sizes vary widely. The virus can escape a broad antibody to hemagglutinin's receptor-binding site the same way it escapes narrow strain-specific antibodies: via single mutations with huge effects. In contrast, broad antibodies to hemagglutinin's stalk only select mutations with small effects. Therefore, among the antibodies we examine, breadth is an imperfect indicator of the potential for viral escape via single mutations. Antibodies targeting the H1 hemagglutinin stalk are quantifiably harder to escape than the other antibodies tested here.
The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed ...vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms—mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant—looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
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•SARS-CoV-2 mRNA vaccines elicit potent GC B cell responses•GC responses are associated with a robust development of neutralizing antibodies•SARS-CoV-2 mRNA vaccines promote antigen-specific Tfh cells•Key elements of the Tfh cell program are modulated by SARS-CoV-2 mRNA vaccines
Herein, Lederer et al. show a nucleic-acid-based vaccine platform for SARS-CoV-2 that potently induces germinal center (GC) responses. GCs are microanatomical sites harboring the formation of high-quality, protective antibody responses. Such vaccine platforms can be promising candidates to mitigate the COVID-19 pandemic.
Many enzymes catalyse reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates
. These enzymes include serine hydrolases
(encoded by one per cent of human genes, and ...including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery
. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours
. In some cases, acyl-enzyme complexes can be stabilized using substrate analogues or active-site mutations but, although these approaches can provide valuable insight
, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code
. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase domain of valinomycin synthetase
, we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.
Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine ...CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13
follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.
We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial ...linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMabCH¹â»CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.
The rhLIF is widely used as an essential factor in stem cell cultures for cell therapies. However, all the recombinant LIFs commercially available are expensive, and no commercially available rhLIF ...meet the standards recommended by USP for use in cell therapies. The current study reports the efficient production of N-glycosylated and bioactive rhLIF in CHO cells. The production rate of established rhLIF-expressing rCHO cells was approximately 0.85 g/l in 12-day fed-batch cultures using a 7.5 l bioreactor. The rhLIF protein was purified via a four-step purification procedure with approximately 57% recovery rate and greater than 99% purity. The purified rhLIF was N-glycosylated and biologically active with an EC50 of 0.167 ng/ml and a specific activity of 0.86 × 103 IU/mg. The purification procedure controlled the levels of process-related impurities below critical levels recommended by USP for cytokines used in cell therapies. The current work is the first production process of N-glycosylated and bioactive rhLIF, which can be applied to large-scale manufacture of GMP-grade rhLIF for use as an ancillary material in cell therapy.
•Stable CHO cell line producing recombinant human LIF (rhLIF) was established.•Establishment of bioreactor culture and purification processes that efficiently produce rhLIF.•The purified rhLIF was N-glycosylated and bioactive.•All process impurity levels satisfy the recommendations of USP for cytokines used in cell therapy.•The production process developed is applicable for scalable GMP manufacturing.
Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant ...protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5–9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.
•A novel, robust protein cage, encapsulin EncMh, was discovered and its structure was elucidated.•High expression of EncMh in Escherichia coli can be achieved.•Various cofactor-containing enzymes can be packaged in encapsulin EncMh.•Enzymes in encapsulin EncMh are more thermostable.•The pores of encapsulin, through a size-exclusion effect, can modulate the substrate profile of an encapsulated enzyme.
Class 2 CRISPR-Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR-Cas constitutes ~60% of all the CRISPR-Cas systems. However, only ...type I-B and I-E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type I-F system to regulate human gene expression. By fusing transcription activation domain to Pseudomonas aeruginosa type I-F Cas proteins, we activate gene transcription in human cells. In most cases, type I-F system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a crRNA array. Furthermore, type I-F system activates target genes specifically without off-target transcription activation. These data demonstrate the robustness and programmability of type I-F CRISPR-Cas in human cells.
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