The physiological landscape and specificity of antibody repertoires are consolidated by multiple immunizations.

Diverse antibody repertoires spanning multiple lymphoid organs (i.e., bone marrow, spleen, lymph nodes) form the foundation of protective humoral immunity.

Accelerating covalent binding studies: Direct mass shift measurement with acoustic ejection and TOF-MS.

Tracking chemical reactions by measuring incurred mass shifts upon successful binding is a direct and attractive alternative to existing assays based on chemical tags. Traditional methods use liquid chromatography-mass spectrometry (LC-MS), and because the required buffers are not amenable to direct MS injection, sample pre-treatment is needed to desalt. This leads to analysis times from ten seconds to minutes per sample, limiting throughput and preventing widespread application.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display.

mRNA Display Identifies Potent, Paralog-Selective Peptidic Ligands for ARID1B.

The ARID1A and ARID1B subunits are mutually exclusive components of the BAF variant of SWI/SNF chromatin remodeling complexes. Loss of function mutations in ARID1A are frequently observed in various cancers, resulting in a dependency on the paralog ARID1B for cancer cell proliferation. However, ARID1B has never been targeted directly, and the high degree of sequence similarity to ARID1A poses a challenge for the development of selective binders.

Protein destabilization underlies pathogenic missense mutations in ARID1B.

ARID1B is a SWI/SNF subunit frequently mutated in human Coffin-Siris syndrome (CSS) and it is necessary for proliferation of ARID1A mutant cancers. While most CSS ARID1B aberrations introduce frameshifts or stop codons, the functional consequence of missense mutations found in ARID1B is unclear. We here perform saturated mutagenesis screens on ARID1B and demonstrate that protein destabilization is the main mechanism associated with pathogenic missense mutations in patients with Coffin-Siris Syndrome.

Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications.

Proteins regulate biological processes by changing their structure or abundance to accomplish a specific function. In response to a perturbation, protein structure may be altered by various molecular events, such as post-translational modifications, protein-protein interactions, aggregation, allostery or binding to other molecules. The ability to probe these structural changes in thousands of proteins simultaneously in cells or tissues can provide valuable information about the functional state of biological processes and pathways.

A generic framework for hierarchical de novo protein design.

De novo protein design enables the exploration of novel sequences and structures absent from the natural protein universe. De novo design also stands as a stringent test for our understanding of the underlying physical principles of protein folding and may lead to the development of proteins with unmatched functional characteristics. The first fundamental challenge of de novo design is to devise "designable" structural templates leading to sequences that will adopt the predicted fold.

Global, in situ analysis of the structural proteome in individuals with Parkinson's disease to identify a new class of biomarker.

Parkinson's disease (PD) is a prevalent neurodegenerative disease for which robust biomarkers are needed. Because protein structure reflects function, we tested whether global, in situ analysis of protein structural changes provides insight into PD pathophysiology and could inform a new concept of structural disease biomarkers. Using limited proteolysis-mass spectrometry (LiP-MS), we identified 76 structurally altered proteins in cerebrospinal fluid (CSF) of individuals with PD relative to healthy donors.

Bottom-up de novo design of functional proteins with complex structural features.

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs.

De novo protein design enables the precise induction of RSV-neutralizing antibodies.

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines.

rstoolbox - a Python library for large-scale analysis of computational protein design data and structural bioinformatics.

Background: Large-scale datasets of protein structures and sequences are becoming ubiquitous in many domains of biological research. Experimental approaches and computational modelling methods are generating biological data at an unprecedented rate. The detailed analysis of structure-sequence relationships is critical to unveil governing principles of protein folding, stability and function.

Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen.

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g.

Rosetta FunFolDes - A general framework for the computational design of functional proteins.

The robust computational design of functional proteins has the potential to deeply impact translational research and broaden our understanding of the determinants of protein function and stability. The low success rates of computational design protocols and the extensive in vitro optimization often required, highlight the challenge of designing proteins that perform essential biochemical functions, such as binding or catalysis. One of the most simplistic approaches for the design of function is to adopt functional motifs in naturally occurring proteins and transplant them to computationally designed proteins.

Structure-based immunogen design-leading the way to the new age of precision vaccines.

Vaccines have been one of the most successful interventions in global health. However, traditional vaccine development has proven insufficient to deal with pathogens that elude the immune system through highly variable and non-functional epitopes. Emerging B cell technologies have yielded potent monoclonal antibodies targeting conserved epitopes, and their structural characterization has provided templates for rational immunogen design.

Correction: Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design.

[This corrects the article DOI: 10.1371/journal.ppat.

Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown.

Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design.

An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features.

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses.