Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.

Src homology 2 domain containing protein 5 (SH2D5) binds the breakpoint cluster region protein, BCR, and regulates levels of Rac1-GTP.

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.

Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy.

Formulation development of a stable influenza recombinant neuraminidase vaccine candidate.

Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi.

Tudor domains are protein modules that mediate protein-protein interactions, potentially by binding to methylated ligands. A group of germline specific single and multiTudor domain containing proteins (TDRDs) represented by drosophila Tudor and its mammalian orthologs Tdrd1, Tdrd4/RNF17, and Tdrd6 play evolutionarily conserved roles in germinal granule/nuage formation and germ cell specification and differentiation. However, their physiological ligands, and the biochemical and structural basis for ligand recognition, are largely unclear. Here, by immunoprecipitation of endogenous murine Piwi proteins (Miwi and Mili) and proteomic analysis of complexes related to the piRNA pathway, we show that the TDRD group of Tudor proteins are physiological binding partners of Piwi family proteins. In addition, mass spectrometry indicates that arginine residues in RG repeats at the N-termini of Miwi and Mili are methylated in vivo. Notably, we found that Tdrkh/Tdrd2, a novel single Tudor domain containing protein identified in the Miwi complex, is expressed in the cytoplasm of male germ cells and directly associates with Miwi. Mutagenesis studies mapped the Miwi-Tdrkh interaction to the very N-terminal RG/RA repeats of Miwi and showed that the Tdrkh Tudor domain is critical for binding. Furthermore, we have solved the crystal structure of the Tdrkh Tudor domain, which revealed an aromatic binding pocket and negatively charged binding surface appropriate for accommodating methylated arginine. Our findings identify a methylation-directed protein interaction mechanism in germ cells mediated by germline Tudor domains and methylated Piwi family proteins, and suggest a complex mode of regulating the organization and function of Piwi proteins in piRNA silencing pathways.

Epitope screening using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): An accelerated workflow for evaluation of lead monoclonal antibodies.

BACKGROUND: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production have enabled candidate mAbs to be identified at a rapidly increasing rate, resulting in a significant bottleneck in the characterization of "structural" epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, a Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen. MAIN METHODS AND MAJOR RESULTS: The method is demonstrated on set of six candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes were unambiguously determined using two HDX mixing timepoints in 24 h total run time, which is equivalent to the instrument time required to map a single epitope using the conventional workflow. CONCLUSION: An accelerated HDX-MS epitope screening workflow was developed. The "screening" workflow successfully characterized five (out of six attempted) novel epitopes on the PRN antigen; information that can be used to support vaccine antigenicity assays.

Protein Footprinting, Conformational Dynamics, and Core Interface-Adjacent Neutralization "Hotspots" in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human ACE2 Interaction.

The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these "binding-site adjacent" dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these "peripheral" sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization "hotspots" outside of the core binding interfaces defined by high resolution structural data.

Immunocartography: Charting vaccine-driven immunity by applying single cell proteomics to an in vitro human model.

The ability to measure immunomodulatory effects of a vaccine is crucial for novel vaccine design. While traditional animal models have been effective, a better understanding of the response in humans to new vaccines in pre-clinical development is critical for advancement to clinical trials. A translational methodology that can capture the complexity of a vaccine-driven response in a human model, which does not require human exposure, is needed. Here we have designed a platform that uses fresh human whole blood as a key component to study the adaptive immune memory response to vaccine formulations. The response is monitored by high-parameter single cell analysis using mass cytometry (Helios, CyTOF System), allowing for a rapid, in-depth characterization of antigen specific proliferation and expansion of preexisting memory T cells in concert with an innate adjuvant-driven response. In this work we demonstrate the capability of this platform to characterize biologically relevant changes in the cellular response across memory T-cells, B cells, monocytes, and NK cells, at an unprecedented level of detail. This approach that we call Immunocartography has the potential to transform the way new vaccines can be assessed before and throughout clinical development.

Design of a Quantitative LC-MS Method for Residual Toxins Adenylate Cyclase Toxin (ACT), Dermonecrotic Toxin (DNT) and Tracheal Cytotoxin (TCT) in Bordetella pertussis Vaccines.

The antigens for acellular pertussis vaccines are made up of protein components that are purified directly from Bordetella pertussis (B. pertussis) bacterial fermentation. As such, there are additional B. pertussis toxins that must be monitored as residuals during process optimization. This paper describes a liquid chromatography mass spectrometry (LC-MS) method for simultaneous analysis of residual protein toxins adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), as well as a small molecule glycopeptide, tracheal cytotoxin (TCT) in a Pertussis toxin vaccine antigen. A targeted LC-MS technique called multiple reaction monitoring (MRM) is used for quantitation of ACT and TCT, which have established limits in drug product formulations. However, DNT is currently monitored in an animal test, which does not have an established quantitative threshold. New approaches for DNT testing are discussed, including a novel standard based on concatenated quantitation sequences for ACT and DNT. Collectively, the method represents a "3-in-1" analytical simplification for monitoring process-related residuals during development of B. pertussis vaccines.

Development of a targeted nanoLC-MS/MS method for quantitation of residual toxins from Bordetella pertussis.

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis) infection. Pertussis pathogenesis is driven by cell-surface adhesion proteins and secreted toxins; some of which have been harnessed for their immunogenic properties as purified antigen components in acellular vaccines. Two of these virulence factors, adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), are protein toxins with potential for co-purification, and therefore must be monitored as process-related impurities during the development of acellular Pertussis vaccine candidates. Here we describe the development of a targeted nanoLC-MS/MS method for absolute quantitation of ACT and DNT in process intermediates from acellular Pertussis antigen purification. Starting from an in silico digest of the toxin sequences, a synthetic peptide screening approach was applied to systematically evaluate candidate sequences as surrogates for protein quantitation. Following refinement to a subset of sequences, absolutely quantified heavy-labelled (AQUA) peptides were implemented in a parallel reaction monitoring (PRM) workflow with limits of detection (LOD) and quantitation (LOQ) in the 12.5-25 amol (2-4 ng/mL) range on-column. In this work, we highlight a 'standards-driven' approach to surrogate peptide selection for protein quantitation. This strategy can be broadly applied in the absence of purified reference material and accelerate quantitative LC-MS method development across multiple sample matrices.

Detection of Residual Proteins UL5 and UL29 Using a Targeted Proteomics Approach in HSV529, a Replication-Deficient HSV-2 Vaccine Candidate.

HSV529 is a replication defective human herpes simplex virus (HSV)-2 viral vaccine candidate in clinical development. An engineered cell line is required to support production of HSV529 by transgenic expression of the HSV-1 transcription factors UL5 (HELI) and UL29 (DNBI). These 2 genes have been deleted from the vaccine candidate to ensure replication deficiency, and the transgene products are thus impurities that must be monitored in the final product. Multiple reaction monitoring (MRM) is a mass spectrometry (MS) workflow that can be used to quickly develop targeted protein detection and quantitation methods. An MRM method was developed for detection of the HSV-1 proteins UL5 and UL29 based on results from nano-liquid chromatography-MS/MS protein analysis of HSV529 material. Sensitivity, specificity, and linearity of response for the MRM workflow were established using high-flow ultra-performance liquid chromatography coupled to a tandem quadrupole mass analyzer. Results show that residual UL5 and UL29 proteins can be detected in the HSV529 candidate, and that MRM analysis provides the appropriate sensitivity and specificity required for quantitation. The transition from nano-flow to ultra-performance driven chromatography was found to improve method robustness without compromising the sensitivity of the assay.

Sample Preparation for Mass Spectrometry Analysis of Protein-Protein Interactions in Cancer Cell Lines and Tissues.

A precisely controlled network of protein-protein interactions constitutes the basis for functional signaling pathways. This equilibrium is more often than not disrupted in cancer cells, by the aberrant expression or activation of oncogenic proteins. Therefore, the analysis of protein interaction networks in cancer cells has become crucial to expand our comprehension of the molecular underpinnings of tumor formation and progression. This protocol describes a sample preparation method for the analysis of signaling complexes by mass spectrometry (MS), following the affinity purification of a protein of interest from a cancer cell line or a solid tumor. In particular, we provide a spin tip-based protease digestion procedure that offers a more rapid and controlled alternative to other gel-based and gel-free methods. This sample preparation protocol represents a useful strategy to identify protein interactions and to gain insight into the molecular mechanisms that contribute to a given cancer phenotype.

Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor.

Signaling pathways are commonly organized through inducible protein-protein interactions, mediated by adaptor proteins that link activated receptors to cytoplasmic effectors. However, we have little quantitative data regarding the kinetics with which such networks assemble and dissolve to generate specific cellular responses. To address this deficiency, we designed a mass spectrometry method, affinity purification-selected reaction monitoring (AP-SRM), which we used to comprehensively and quantitatively investigate changes in protein interactions with GRB2, an adaptor protein that participates in a remarkably diverse set of protein complexes involved in multiple aspects of cellular function. Our data reliably define context-specific and time-dependent networks that form around GRB2 after stimulation, and reveal core and growth factor-selective complexes comprising 90 proteins identified as interacting with GRB2 in HEK293T cells. Capturing a key hub protein and dissecting its interactions by SRM should be equally applicable to quantifying signaling dynamics for a range of hubs in protein interaction networks.

Eukaryotic protein domains as functional units of cellular evolution.

Modular protein domains are functional units that can be modified through the acquisition of new intrinsic activities or by the formation of novel domain combinations, thereby contributing to the evolution of proteins with new biological properties. Here, we assign proteins to groups with related domain compositions and functional properties, termed "domain clubs," which we use to compare multiple eukaryotic proteomes. This analysis shows that different domain types can take distinct evolutionary trajectories, which correlate with the conservation, gain, expansion, or decay of particular biological processes. Evolutionary jumps are associated with a domain that coordinately acquires a new intrinsic function and enters new domain clubs, thereby providing the modified domain with access to a new cellular microenvironment. We also coordinately analyzed the covalent and noncovalent interactions of different domain types to assess the molecular compartment occupied by each domain. This reveals that specific subsets of domains demarcate particular cellular processes, such as growth factor signaling, chromatin remodeling, apoptotic and inflammatory responses, or vesicular trafficking. We suggest that domains, and the proteins in which they reside, are selected during evolution through reciprocal interactions with protein domains in their local microenvironment. Based on this scheme, we propose a mechanism by which Tudor domains may have evolved to support different modes of epigenetic regulation and suggest a role for the germline group of mammalian Tudor domains in Piwi-regulated RNA biology.