Molecular mechanism of contactin 2 homophilic interaction.

Contactin 2 (CNTN2) is a cell adhesion molecule involved in axon guidance, neuronal migration, and fasciculation. The ectodomains of CNTN1-CNTN6 are composed of six Ig domains (Ig1-Ig6) and four FN domains. Here, we show that CNTN2 forms transient homophilic interactions (KD ∼200 nM). Cryo-EM structures of full-length CNTN2 and CNTN2_Ig1-Ig6 reveal a T-shaped homodimer formed by intertwined, parallel monomers. Unexpectedly, the horseshoe-shaped Ig1-Ig4 headpieces extend their Ig2-Ig3 tips outwards on either side of the homodimer, while Ig4, Ig5, Ig6, and the FN domains form a central stalk. Cross-linking mass spectrometry and cell-based binding assays confirm the 3D assembly of the CNTN2 homodimer. The interface mediating homodimer formation differs between CNTNs, as do the homophilic versus heterophilic interaction mechanisms. The CNTN family thus encodes a versatile molecular platform that supports a very diverse portfolio of protein interactions and that can be leveraged to strategically guide neural circuit development.

Microneedle-based sampling of dermal interstitial fluid using a vacuum-assisted skin patch.

Interstitial fluid (ISF) contains a wealth of biomolecules, yet it is underutilized for diagnostic testing due to a lack of rapid and simple techniques for collecting abundant amounts of fluid. Here, we report a simple and minimally invasive technique for rapidly sampling larger quantities of ISF from human skin. A microneedle array is used to generate micropores in skin from which ISF is extracted using a vacuum-assisted skin patch. Using this technique, an average of 20.8 µL of dermal ISF is collected in 25 min, which is an ∼6-fold improvement over existing sampling methods. Proteomic analysis of collected ISF reveals that it has nearly identical protein composition as blood, and >600 medically relevant biomarkers are identified. Toward this end, we demonstrate the detection of SARS-CoV-2 neutralizing antibodies in ISF collected from COVID-19 vaccinees using two commercial immunoassays, showcasing the utility of this technique for diagnostic testing.

Filling the gaps in peptide maps with a platform assay for top-down characterization of purified protein samples.

Liquid chromatography-mass spectrometry (LC-MS) intact mass analysis and LC-MS/MS peptide mapping are decisional assays for developing biological drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We leveraged the strengths of flow-programmed (fp) denaturing online buffer exchange (dOBE) chromatography, including robust automation, relatively high ESI sensitivity, and long MS/MS window time, to support a TDMS platform for industrial protein characterization. We tested data-dependent (DDA) and targeted strategies using 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large, focused dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. A DDA-based workflow provided objective identification of αLac truncation proteoforms with a two-termini clipping search. A targeted TDMS workflow facilitated the characterization of αLac oxidation positional isomers. This strategy relied on using sliding window-based fragment ion deconvolution to generate composite proteoform spectral match (cPrSM) results amenable to fragment noise filtering, which is a fundamental enhancement relevant to TDMS applications generally.

Decriminalization or police mission creep? Critical appraisal of law enforcement involvement in British Columbia, Canada's decriminalization framework.

The unregulated drug toxicity crisis in British Columbia (BC), Canada, has claimed over 14,000 lives since 2016. The crisis is shaped by prohibitionist policies that has led to the contamination of the unregulated drug supply, resulting in a surge of fatal and non-fatal overdose events. The criminalization of drug users exacerbates this situation, pushing individuals into carceral systems for the possession of and/or social practices related to drug use. This commentary examines the involvement of policing in the development, and throughout the first 15 months of its implementation, of BC's decriminalization framework. We highlight concerns regarding police discretion, the expansion of scope, and the interweaving of carceral logics into policies that purport to be public health-oriented.

NCCN Policy Summit: Cancer Across Geography.

Geographic location of a patient directly impacts access to care, including preventive screenings and early detection. Although there is a higher prevalence of the most common cancers in urban areas, mortality rates are higher in rural communities. Notably, indigenous communities residing on tribal lands often experience heightened access issues and environmental exposure to known and probable human carcinogens. The burdens associated with a cancer diagnosis can be exacerbated by various barriers to accessing quality care; however, there are emerging best practices to overcome these barriers. Understanding the interplay between geography and a patient's access to cancer care services is crucial for addressing existing disparities and ensuring equitable health care provision across regions. By leveraging innovative policy and practice solutions, communities can begin to close care gaps and establish bidirectional trust between patients and providers across the care continuum, which is necessary to enact meaningful reforms. To advance the conversation on geographic disparities and strategies that mitigate associated barriers to care, NCCN hosted the Policy Summit "Cancer Across Geography" on June 15, 2023, at the National Press Club in Washington, DC. Through keynote addresses and multistakeholder panel discussions, this hybrid event explored care imbalances across geography, recent policy and technology advancements, and current challenges associated with cancer care. This created a forum for a diverse group of attendees to thoughtfully discuss policies and practices to advance high-quality, effective, efficient, equitable, and accessible cancer care for all. Speakers and attendees featured multidisciplinary clinicians, epidemiologists, community oncologists, researchers, payers, patient advocates, industry, providers, policymakers, and leaders representing underserved communities, among others.

The myosin chaperone UNC-45 has an important role in maintaining the structure and function of muscle sarcomeres during adult aging.

C. elegans undergo age-dependent declines in muscle organization and function, similar to human sarcopenia. The chaperone UNC-45 is required to fold myosin heads after translation and is likely used for refolding after thermally- or chemically-induced unfolding. UNC-45's TPR region binds HSP-90 and its UCS domain binds myosin heads. We observe early onset sarcopenia when UNC-45 is reduced at the beginning of adulthood. There is sequential decline of HSP-90, UNC-45, and MHC B myosin. A mutation in age-1 delays sarcopenia and loss of HSP-90, UNC-45, and myosin. UNC-45 undergoes age-dependent phosphorylation, and mass spectrometry reveals phosphorylation of six serines and two threonines, seven of which occur in the UCS domain. Additional expression of UNC-45 results in maintenance of MHC B myosin and suppression of A-band disorganization in old animals. Our results suggest that increased expression or activity of UNC-45 might be a strategy for prevention or treatment of sarcopenia.

Inefficient recruitment of DDX39B impedes pre-spliceosome assembly on FOXP3 introns.

Forkhead box P3 (FOXP3) is the master fate-determining transcription factor in regulatory T (Treg) cells and is essential for their development, function, and homeostasis. Mutations in FOXP3 cause immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, and aberrant expression of FOXP3 has been implicated in other diseases such as multiple sclerosis and cancer. We previously demonstrated that pre-mRNA splicing of FOXP3 RNAs is highly sensitive to levels of DExD-box polypeptide 39B (DDX39B), and here we investigate the mechanism of this sensitivity. FOXP3 introns have cytidine (C)-rich/uridine (U)-poor polypyrimidine (py) tracts that are responsible for their inefficient splicing and confer sensitivity to DDX39B. We show that there is a deficiency in the assembly of commitment complexes (CCs) on FOXP3 introns, which is consistent with the lower affinity of U2AF2 for C-rich/U-poor py tracts. Our data indicate an even stronger effect on the conversion of CCs to pre-spliceosomes. We propose that this is due to an altered conformation that U2AF2 adopts when it binds to C-rich/U-poor py tracts and that this conformation has a lower affinity for DDX39B. As a consequence, CCs assembled on FOXP3 introns are defective in recruiting DDX39B, and this leads to the inefficient assembly of pre-spliceosome complexes.

Translating the lived experience of illicit drinkers into program guidance for cannabis substitution: Experiences from the Canadian Managed Alcohol Program Study.

A small but growing body of research has suggested the potential for cannabis substitution to support Managed Alcohol Program (MAP) service users to reduce acute and chronic alcohol-related harms. In 2022, researchers from the Canadian Managed Alcohol Program Study (CMAPS) noted a dearth of accessible, alcohol-specific educational resources to support service users and program staff to implement cannabis substitution pilots at several MAP sites in Canada. In this essay, we draw on over 10-years of collaboration between CMAPS, and organizations of people with lived experience (the Eastside Illicit Drinkers Group for Education (EIDGE) and SOLID Victoria) to describe our experiences co-creating cannabis education resources where none existed to support MAP sites interested in beginning to provide cannabis to participants. The research team relied on the unique lived experiences and informal cannabis-related harm reduction strategies described by EIDGE and SOLID members to create cannabis education resources that were accurate and relevant to MAP sites. EIDGE was familiar with creating peer-oriented educational resources and convened meetings and focus groups to engage peers. CMAPS research team members created standard cannabis unit equivalencies to support program delivery, and clinical advisors ensured that the stated risks and benefits of cannabis substitution, as well as tapering guidance for withdrawal management, were safe and feasible. The collaboration ultimately produced tailored client-facing and provider-facing resources. Our experience demonstrates that the lived expertise of drinkers can play an integral role in creating alcohol harm reduction informational materials, specifically those related to cannabis substitution, when combined with data from rigorous, community-based programs of research like CMAPS. We close by listing additional considerations for cannabis substitution program design for MAP settings emerging from this process of collaboration between illicit drinkers, service providers, clinicians, and researchers for consideration by other programs.

Vancouver's Alcohol Knowledge Exchange: lessons learned from creating a peer-involved alcohol harm reduction strategy in Vancouver's Downtown Eastside.

Despite high rates of harm attributable to alcohol use itself and the associated marginalization of illicit drinkers in Vancouver's Downtown Eastside (DTES), alcohol-specific harm reduction services there are under-resourced and highly disconnected from one another. In response to these conditions and high rates of death amongst its membership, the Eastside Illicit Drinkers Group for Education, an affiliate group of the Vancouver Area Network of Drug Users, convened a regular meeting of stakeholders, termed a "community of practice" in 2019 to bring together peers who used beverage and non-beverage alcohol, shelter and harm reduction service providers, public health professionals, clinicians, and policymakers to improve system-level capacity to reduce alcohol-related harm. The discussions that followed from these meetings were transformed into the Vancouver Alcohol Strategy (VAS), a comprehensive, harm reduction-oriented policy framework for alcohol harm reduction in the DTES. This article highlights our experiences producing community-led alcohol policy through the VAS with specific attention to the ways in which people who use alcohol themselves were centred throughout the policy development process. We also provide summary overviews of each of the VAS document's 6 thematic areas for action, highlighting a sampling of the 47 total unique recommendations. Historically, people who use non-beverage alcohol and whose use of alcohol in public spaces is criminalized due to housing precarity and visible poverty have been excluded from the development of population-level alcohol policies that can harm this specific population. The process of policy development undertaken by the VAS has attempted to resist this top-down approach to public health policy development related to alcohol control by intentionally creating space for people with lived experience to guide our recommendations. We conclude by suggesting that a grassroots enthusiasm for harm reduction focused policy development exists in Vancouver's DTES, and requires resources from governmental public health institutions to meaningfully prevent and reduce alcohol-related and policy-induced harms.

Designer molecules of the synaptic organizer MDGA1 reveal 3D conformational control of biological function.

MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors) are synaptic cell surface molecules that regulate the formation of trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs), which promote synaptic development. Mutations in MDGAs are implicated in various neuropsychiatric diseases. MDGAs bind NLGNs in cis on the postsynaptic membrane and physically block NLGNs from binding to NRXNs. In crystal structures, the six immunoglobulin (Ig) and single fibronectin III domains of MDGA1 reveal a striking compact, triangular shape, both alone and in complex with NLGNs. Whether this unusual domain arrangement is required for biological function or other arrangements occur with different functional outcomes is unknown. Here, we show that WT MDGA1 can adopt both compact and extended 3D conformations that bind NLGN2. Designer mutants targeting strategic molecular elbows in MDGA1 alter the distribution of 3D conformations while leaving the binding affinity between soluble ectodomains of MDGA1 and NLGN2 intact. In contrast, in a cellular context, these mutants result in unique combinations of functional consequences, including altered binding to NLGN2, decreased capacity to conceal NLGN2 from NRXN1ß, and/or suppressed NLGN2-mediated inhibitory presynaptic differentiation, despite the mutations being located far from the MDGA1-NLGN2 interaction site. Thus, the 3D conformation of the entire MDGA1 ectodomain appears critical for its function, and its NLGN-binding site on Ig1-Ig2 is not independent of the rest of the molecule. As a result, global 3D conformational changes to the MDGA1 ectodomain via strategic elbows may form a molecular mechanism to regulate MDGA1 action within the synaptic cleft.

DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein.

Neutrophil extracellular traps (NETs) are produced through ejection of genomic DNA by neutrophils into extracellular space and serve as a weapon to fight against pathogens. Neutrophil elastase, a serine protease loaded on NETs, attacks and kills pathogens, while extracellular high-mobility-group-box-1 (HMGB1) protein serves as a danger signal to other cells. How the action of these factors is coordinated as part of the innate immune response is not fully understood. In this article, using biochemical and biophysical approaches, we demonstrate that DNA mediates specific proteolysis of HMGB1 by neutrophil elastase and that the proteolytic processing remarkably enhances binding activities of extracellular HMGB1. Through the DNA-mediated proteolysis of HMGB1 by neutrophil elastase, the negatively charged segment containing D/E repeats is removed from HMGB1. This proteolytic removal of the C-terminal tail causes a substantial increase in binding activities of HMGB1 because the D/E repeats are crucial for dynamic autoinhibition via electrostatic interactions. Our data on the oxidized HMGB1 (i.e., 'disulfide HMGB1') protein show that the truncation substantially increases HMGB1's affinities for the toll-like receptor TLR4•MD-2 complex, DNA G-quadruplex, and the Holliday junction DNA structure. The DNA-mediated proteolysis of HMGB1 by neutrophil elastase in NETs may promote the function of extracellular HMGB1 as a damage-associated molecular pattern that triggers the innate immune response of nearby cells.

Comparative genomic and biochemical analyses identify a collagen galactosylhydroxylysyl glucosyltransferase from Acanthamoeba polyphaga mimivirus.

Humans and Acanthamoeba polyphaga mimivirus share numerous homologous genes, including collagens and collagen-modifying enzymes. To explore this homology, we performed a genome-wide comparison between human and mimivirus using DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) and identified 52 new putative mimiviral proteins that are homologous with human proteins. To gain functional insights into mimiviral proteins, their human protein homologs were organized into Gene Ontology (GO) and REACTOME pathways to build a functional network. Collagen and collagen-modifying enzymes form the largest subnetwork with most nodes. Further analysis of this subnetwork identified a putative collagen glycosyltransferase R699. Protein expression test suggested that R699 is highly expressed in Escherichia coli, unlike the human collagen-modifying enzymes. Enzymatic activity assay and mass spectrometric analyses showed that R699 catalyzes the glucosylation of galactosylhydroxylysine to glucosylgalactosylhydroxylysine on collagen using uridine diphosphate glucose (UDP-glucose) but no other UDP-sugars as a sugar donor, suggesting R699 is a mimiviral collagen galactosylhydroxylysyl glucosyltransferase (GGT). To facilitate further analysis of human and mimiviral homologous proteins, we presented an interactive and searchable genome-wide comparison website for quickly browsing human and Acanthamoeba polyphaga mimivirus homologs, which is available at RRID Resource ID: SCR_022140 or https://guolab.shinyapps.io/app-mimivirus-publication/ .

Chemically targeting the redox switch in AP1 transcription factor ΔFOSB.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.

QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.

Esomeprazole covalently interacts with the cardiovascular enzyme dimethylarginine dimethylaminohydrolase: Insights into the cardiovascular risk of proton pump inhibitors.

BACKGROUND: Proton pump inhibitors (PPIs) are widely prescribed drugs for the treatment of gastroesophageal reflux disease (GERD). Several meta-analysis studies have reported associations between prolonged use of PPIs and major adverse cardiovascular events. However, interaction of PPIs with biological molecules involved in cardiovascular health is incompletely characterized. Dimethylarginine dimethylaminohydrolase (DDAH) is a cardiovascular enzyme expressed in cardiomyocytes, and other somatic cell types in one of two isotypes (DDAH1 and DDAH2) to metabolize asymmetric dimethylarginine (ADMA); a cardiovascular risk factor and competitive inhibitor of nitric oxide synthases (NOSs). METHODS: We performed high throughput drug screening of over 130,000 small molecules to discover human DDAH1 inhibitors and found that PPIs directly inhibit DDAH1. We expressed and purified the enzyme for structural and mass spectrometry proteomics studies to understand how a prototype PPI, esomeprazole, interacts with DDAH1. We also performed molecular docking studies to model the interaction of DDAH1 with esomeprazole. X-ray crystallography was used to determine the structure of DDAH1 alone and bound to esomeprazole at resolutions ranging from 1.6 to 2.9 Å. RESULTS: Analysis of the enzyme active site shows that esomeprazole interacts with the active site cysteine (Cys273) of DDAH1. The structural studies were corroborated by mass spectrometry which indicated that cysteine was targeted by esomeprazole to inactivate DDAH1. CONCLUSIONS: The inhibition of this important cardiovascular enzyme by a PPI may help explain the reported association of PPI use and increased cardiovascular risk in patients and the general population. GENERAL SIGNIFICANCE: Our study calls for pharmacovigilance studies to monitor adverse cardiovascular events in chronic PPI users.

AhR promotes phosphorylation of ARNT isoform 1 in human T cell malignancies as a switch for optimal AhR activity.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform­specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform­specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders.

Ion-Ion Charge Reduction Addresses Multiple Challenges Common to Denaturing Intact Mass Analysis.

Complete LC-MS-based protein primary sequence characterization requires measurement of intact protein profiles under denaturing and/or reducing conditions. To address issues of protein overcharging of unstructured proteins under acidic, denaturing conditions and sample heterogeneity (macro- and micro-scales) which often confound denaturing intact mass analysis of a wide variety of protein samples, we propose the use of broadband isolation of entire charge state distributions of intact proteins followed by ion-ion proton transfer charge reduction, which we have termed "full scan PTCR" (fsPTCR). Using rapid denaturing size exclusion chromatography coupled to fsPTCR-Orbitrap MS and time-resolved deconvolution data analysis, we demonstrate a strategy for method optimization, leading to significant analytical advantages over conventional MS1. Denaturing analysis of the flexible bacterial translation initiation factor 2 (91 kDa) using fsPTCR reduced overcharging and showed an 11-fold gain in S/N compared to conventional MS1. Analysis by fsPTCR-MS of the microheterogeneous glycoprotein fetuin revealed twice as many proteoforms as MS1 (112 vs 56). In a macroheterogeneous mixture of proteins ranging from 14 to 148 kDa, fsPTCR provided more than 10-fold increased sensitivity and quantitative accuracy for diluted bovine serum albumin (66 kDa). Finally, our analysis shows that collisional gas pressure is a key parameter which can be utilized during fsPTCR to retain or remove larger proteins from acquired spectra.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.