Affinity maturation

The viral vector-based COVID-19 vaccine Ad26.COV2.S has been recommended by the WHO since 2021 and has been administered to over 200 million people. Prior studies have shown that Ad26.COV2.S induces durable neutralizing antibodies (NAbs) that increase in coverage of variants over time, even in the absence of boosting or infection. Here, we studied humoral responses following Ad26.COV2.S vaccination in individuals enrolled in the initial Phase 1/2a trial of Ad26.COV2.S in 2020. Through 8 months post vaccination, serum NAb responses increased to variants, including B...

B cell transcriptomic signatures hold promise for the early prediction of vaccine-induced humoral immunity and vaccine protective efficacy. We performed a longitudinal study in 232 healthy adult participants before/after a 3rd dose of MMR (MMR3) vaccine. We assessed baseline and early transcriptional patterns in purified B cells and their association with measles-specific humoral immunity after MMR vaccination using two analytical methods ("per gene" linear models and joint analysis). Our study identified distinct early transcriptional signatures/genes following MMR3 that were associated with measles-specific neutralizing antibody titer and/or binding antibody titer...

BACKGROUND: Radionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies...

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) by introducing base substitutions into antibody genes, a process enabling antibody affinity maturation in immune response. How a mutator is tamed to precisely and safely generate programmed DNA lesions in a physiological process remains unsettled, as its dysregulation drives lymphomagenesis. Recent research has revealed several hidden features of AID-initiated mutagenesis: preferential activity on flexible DNA substrates, restrained activity within chromatin loop domains, unique DNA repair factors to differentially decode AID-caused lesions, and diverse consequences of aberrant deamination...

ProNGF (nerve growth factor) is a precursor of NGF and a signaling peptide exerting opposite effects on neuronal cells, i.e., apoptotic or neuritogenic. The conflicting biological activity of proNGF depends on the relative levels of two membrane receptors, TrkA and p75NTR. The effect of proNGF depends on the expression levels of these receptor proteins and their affinity to proNGF. Since the affinity of proteins has been studied with various recombinant proteins, it is worth comparing the affinity of these proteins within one experiment with the same method...

COVID-19 induces re-circulating long-lived memory B cells (MBC) that, upon re-encounter with the pathogen, are induced to mount immunoglobulin responses. During convalescence, antibodies are subjected to affinity maturation, which enhances the antibody binding strength and generates new specificities that neutralize virus variants. Here, we performed a single-cell RNA sequencing analysis of spike-specific B cells from a SARS-CoV-2 convalescent subject. After COVID-19 vaccination, matured infection-induced MBC underwent recall and differentiated into plasmablasts...

Stent implantation is one of the most effective methods for the treatment of atherosclerosis. Nitinol stent is a type of stent with good biocompatibility and relatively mature development; however, it cannot effectively achieve long-term anticoagulation and early endothelialization. In this study, nitinol surfaces with the programmed assembly of heparin, exosomes from endothelial cells, and endothelial affinity peptide (REDV) were fabricated through layer-by-layer assembly technology and click-chemistry, and then exosomes/REDV-modified nitinol interface (ACC-Exo-REDV) was prepared...

The on-going diversification of influenza virus necessicates annual vaccine updating. The vaccine antigen, the viral spike protein hemagglutinin (HA), tends to elicit strain-specific neutralizing activity, predicting that sequential immunization with the same HA strain will boost antibodies with narrow coverage. However, repeated vaccination with homologous SARS-CoV-2 vaccine eventually elicits neutralizing activity against highly unmatched variants, questioning this immunological premise. We evaluated a longitudinal influenza vaccine cohort, where each year the subjects received the same, novel H1N1 2009 pandemic vaccine strain...

During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3), members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined...

Nipah virus (NiV) is a World Health Organization priority pathogen and there are currently no approved drugs for clinical immunotherapy. Through the use of a naïve human phage-displayed Fab library, two neutralizing antibodies (NiV41 and NiV42) targeting the NiV receptor binding protein (RBP) were identified. Following affinity maturation, antibodies derived from NiV41 display cross-reactivity against both NiV and Hendra virus (HeV), whereas the antibody based on NiV42 is only specific to NiV. Results of immunogenetic analysis reveal a correlation between the maturation of antibodies and their antiviral activity...

The transition from immunoglobulin M (IgM) to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential maintenance of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different Ig isotypes within GCs is dependent on specific transcriptional mechanisms. Here, we explored IgG1+ GC B cell transcription factor dependency using a CRISPR-Cas9 screen and conditional mouse genetics...

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa . The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending...

Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions...

The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion...

An initial virus exposure can imprint antibodies such that future responses to antigenically drifted strains are dependent on the identity of the imprinting strain. Subsequent exposure to antigenically distinct strains followed by affinity maturation can guide immune responses toward generation of cross-reactive antibodies. How viruses evolve in turn to escape these imprinted broad antibody responses is unclear. Here, we used clonal antibody lineages from two human donors recognizing conserved influenza virus hemagglutinin (HA) epitopes to assess viral escape potential using deep mutational scanning...

Antibody affinity maturation occurs in secondary lymphoid organs within germinal centers (GCs). At these sites, B cells mutate their antibody-encoding genes in the dark zone, followed by preferential selection of the high-affinity variants in the light zone by T cells. The strength of the T cell-derived selection signals is proportional to the B cell receptor affinity and to the magnitude of subsequent Myc expression. However, because the lifetime of Myc mRNA and its corresponding protein is very short, it remains unclear how T cells induce sustained Myc levels in positively selected B cells...

Antigen-specific antibody infusion is known to enhance or suppress germinal center (GC) responses depending on the affinity of the infusion. We hypothesized that infusing monoclonal antibodies (mAbs) of escalating affinity during an immunization regimen may progressively escalate selection pressure on competing B cells, increasing their affinity. To test this, we immunized mice with HIV envelope gp120 and infused CD4 binding-site (CD4bs)-specific mAbs. While mAb infusion reduced somatic hypermutation (SHM) and affinity in most CD4bs-specific B cells, a sub-population was identified with greater SHM and affinity than control...

Peptidomic techniques are powerful tools to identify peptides in a biological sample. In the case of brain, which contains a complex mixture of cell types, standard peptidomics procedures reveal the major peptides in a dissected brain region. It is difficult to obtain information on peptides within a specific cell type using standard approaches, unless that cell type can be isolated. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells...

Melanocyte differentiation is orchestrated by the master regulator transcription factor MITF. However, its ability to discern distinct binding sites linked to effective gene regulation remains poorly understood. This study aims to assess how co-activator acetyltransferase interacts with MITF to modulate their related lysine action, thereby mediating downstream gene regulation, including DNA affinity, stability, transcriptional activity, particularly in the process of shell pigmentation. Here, we have demonstrated that the CgMITF protein can be acetylated, further enabling selective amplification of the melanocyte maturation program...

Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology...