Biology subjects

Chen, W.-H.

Publications and source records attributed to Chen, W.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineering a performance-improved, axon-targeted kalium channelrhodopsin for optogenetic neuropathway inhibition

Optogenetic neuropathway inhibition is a powerful approach for dissecting circuit functions. This strategy, however, frequently encounters practical challenges due to insufficient expression or performance of the optogenetic silencer on axonal projections/terminals. HcKCR1, a light-gated potassium-selective channel from Hyphochytrium catenoides, has shown great promise for optogenetic inhibition. Unfortunately, the application of HcKCR1 in neuropathway manipulations is hindered by its unsatisfactory gating properties and poor axonal trafficking. To overcome these hurdles, we first engineered a performance-improved HcKCR1 (piKCR) that allowed more reliable neuronal inhibition at low intensities of green or red light. We next engineered an axon-targeted piKCR (piKCR.AT) that demonstrated long-range axonal trafficking and optical presynaptic inhibition in the mouse hippocampus. When piKCR.AT was expressed in the cerebellar Purkinje Cells (PCs), optical manipulation of PC outputs to the deep cerebellar nuclei robustly disrupted mouse movement on the balance beam. With enhanced performance and axonal distribution, piKCR.AT may provide new opportunities for elucidating neuropathway functions in health and diseases.

bioengineering↗

bioRxiv2026

Develop a durable, memory-driven, CspZ-targeting Lyme disease vaccine by rationale adjuvant selection

Rational adjuvant selection is a systematic approach based on adjuvant-mediated immunomodulation to identify safe vaccine regimens that enhance protective immunity. Transmitted through ticks and caused by the bacterium Borrelia burgdorferi (Bb), Lyme disease (LD) is the most common vector-borne disease in the Northern hemisphere. There are no effective vaccines, making it suitable for testing the concept of rational adjuvant selection. Here, we formulated our previously developed and effective LD vaccine antigen, CspZ-YAC187S, with different adjuvants suitable for human use; we analyzed the immune response by transcriptomics and tested the vaccine efficacy after Bb infection. We identified Alum-CpG and Alum-Gal to elicit the highest titers of CspZ-YAC187S-dependent protective antibodies and robust levels of protection but through distinct mechanisms of immunomodulation. We demonstrated that immunization with Alum-CpG formulated CspZ-YAC187S provided up to nine months of protective bactericidal antibody titers, as well as recall-memory response to prevent LD after natural infection. Immunity was linked to elevated levels of IgG1 memory cells in the vaccine-triggered immune responses. This work thus identified a durable, memory immunity-driven LD vaccine, ultimately paving the road to understanding the mechanisms of rationale adjuvant selection for vaccine development.

immunology↗

bioRxiv2025

A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing

Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present "simplified 5PSeq" (s5PSeq), a streamlined protocol for profiling 5 monophosphorylated (5P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition--offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under four hours and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3000 reads. In addition to simplifying the study of 5P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics. HighlightsO_LIs5PSeq is a streamlined protocol for profiling 5P mRNA degradation intermediates. C_LIO_LIContext-specific ribosome stalls can be used to assess phenotypic antimicrobial sensitivity at the molecular level. C_LIO_LIBlocking rRNA sequencing at the ligation step streamlines library preparation and lowers costs and hands-on time. C_LIO_LIIntegration with nanopore sequencing allows same-day antimicrobial sensitivity testing in species with 5-3 exonuclease. C_LI

microbiology↗

bioRxiv2024

Vaginal Microbiome and Inflammation Among Chinese Women.

Human papillomavirus (HPV) infection, cervical intraepithelial neoplasia (CIN), pelvic organ prolapse (POP), cervical polyps (CP), abnormal uterine bleeding (AUB), and vaginitis are common conditions encountered in gynecological practice. In this study, we collected vaginal secretions from patients diagnosed with these diseases (n = 310) and from healthy donors (n = 112) to analyze their vaginal microbiome using 16S rRNA sequencing and cytokine expression using a cytometric bead array (CBA). Our findings revealed that the vaginal microbiome in POP patients exhibited higher complexity compared to other groups. Regarding cytokine expression, levels of IL-1, IL-1{beta}, IL-6, IL-8, MCP-1, and MIG were significantly elevated in HPV and CIN patients. Analysis of microbial associations showed that Lactobacillus crispatus and Lactobacillus iners were significantly negatively correlated with IL-1 and IL-1{beta} expression. In contrast, non-Lactobacillus bacteria, including Bifidobacterium breve, Prevotella bivia, Gardnerella vaginalis, Sneathia amnii, Sneathia sanguinegens, Prevotella amnii, Escherichia coli, and Chlamydia trachomatis, were positively correlated with IL-1, IL-1{beta}, IL-6, IL-8, MCP-1, and MIG levels. Furthermore, Lactobacillus iners exhibited a significant negative effect in the HPV and CIN patient groups.

microbiology↗

bioRxiv2024

Mechanistic insights into structure-based design of a Lyme disease subunit vaccine

The quality of protective immunity plays a critical role in modulating vaccine efficacy, with native antigens often not able to trigger sufficiently strong immune responses for pathogen killing. This warrants creation of structure-based vaccine design, leveraging high-resolution antigen structures for mutagenesis to improve protein stability and efficient immunization strategies. Here, we investigated the mechanisms underlying structure-based vaccine design using CspZ-YA, a vaccine antigen from Borrelia burgdorferi, the bacteria causing Lyme disease (LD), the most common vector-borne disease in the Northern Hemisphere. Compared to wild-type CspZ-YA, we found CspZ-YAI183Y and CspZ-YAC187S required lower immunization frequency to protect mice from LD-associated manifestations and bacterial colonization. We observed indistinguishable human and mouse antigenicity between wild-type and mutant CspZ-YA proteins after native infection or active immunization. This supports our newly generated, high-resolution structures of CspZ-YAI183Y and CspZ-YAC187S, showing no altered surface epitopes after mutagenesis. However, CspZ-YAI183Y and CspZ-YAC187S favored the interactions between helices H and I, consistent with their elevated thermostability. Such findings are further strengthened by increasing ability of protective CspZ-YA monoclonal antibodies in binding to CspZ-YA at a physiological temperature (37{degrees}C). Overall, this study demonstrated enhanced intramolecular interactions improved long-term stability of antigens while maintaining protective epitopes, providing a mechanism for structure-based vaccine design. These findings can ultimately be extended to other vaccine antigens against newly emerging pathogens for the improvement of protective immunity.

immunology↗

bioRxiv2023

Utilization of Eco-friendly Imidazolium-Based Ionic Liquids for Gluten Extraction: Investigating the Influence of Side Chains and Anions

Gluten is a well-known food allergen globally, capable of triggering immune responses in both celiac and non-celiac gluten-sensitive individuals. Gluten comprises two major proteins: glutenin and gliadin. Gliadin, in particular, possesses a unique hydrophobic amino acid sequence. The Food and Drug Administration asserts that the toxicity of gliadin cannot be eliminated through fermentation and hydrolysis processes. A common approach to addressing gluten allergies is to adopt a gluten-free diet. However, the hydrophobic nature of gluten makes its detection challenging. Analysts often resort to using organic solvents or employing multiple procedures to denature gluten for extraction. Its worth noting that while organic solvents can rapidly extract gluten from a sample, they may also alter antibodies, leading to erroneous bio-test results. Ionic liquid (IL) is a highly adaptable green chemical compound consisting of organic salts. We modified imidazolium, a cationic structure, with various carbon side chain lengths (C=0, 1, 3, 5, 7, 9, and 12), and combined it with organic and inorganic anions (e.g., OMs-, Cl-, F-, NO{not}3-, HSO4-, and H2PO4-). We used different IL-to-water ratios to assess gluten solubility. We measured the solubility of gliadin in various imidazolium ILs and conducted kinetic studies on the dissolution of gliadin in 1% [C5DMIM][OMs]aq. Additionally, circular dichroism (CD) spectroscopy and enzyme-linked immunosorbent assay (ELISA) were employed to evaluate the structural changes in gliadin and its interaction with antibodies after treatment with 1% [C5DMIM][OMs]aq. An XTT assay was conducted to assess the cytotoxicity of [C5DMIM][OMs]aq on N2a cells. Our research findings indicate that 1% [C5DMIM][OMs]aq demonstrated excellent gluten solubility, dissolving more than 3000 ppm of gluten within 5 minutes. Importantly, [C5DMIM][OMs]aq did not disrupt the gluten structure, did not impede antibody binding to gluten, and exhibited no cell toxicity. This report highlights [C5DMIM][OMs] as a promising extraction solution for gluten detection.

biochemistry↗

bioRxiv2022

Enterotypes of the human gut mycobiome

The fungal component of the human gut microbiome, also known as the mycobiome, plays a vital role in intestinal ecology and human health. Here, we identify and characterize four mycobiome enterotypes using ITS profiling of 3,363 samples from 16 cohorts across three continents, including 572 newly profiled samples from China. These enterotypes exhibit stability across populations and geographical locations and significant correlation with bacterial enterotypes. Particularly, we notice that fungal enterotypes have a strong age preference, where the enterotype dominated by Candida (i.e., fun_C_E enterotype) is enriched in the elderly population and confers an increased risk of multiple diseases associated with compromised intestinal barrier. In addition, bidirectional mediation analysis reveals that the fungi-contributed aerobic respiration pathway associated with fun_C_E enterotype might mediate the association between the compromised intestinal barrier and aging. TeaserAs an integral part of the human gut microbiome, the fungi, which co-habit with prokaryotic microbiome in the gut, play important role in the intestinal ecology and human health. Yet, the overall structure of the human gut mycobiome and the inter-individual variation worldwide remain largely unclear. Lai et al. analyzed the fungal profiles of 3,363 samples from 16 cohorts across three continents, and identified four fungal enterotypes that exhibit stability across populations. They found that fungal enterotypes showed age preference, where a Candida dominated enterotype was enriched in the elderly population and confers an increased risk of multiple diseases and more severe compromised intestinal barrier. Furthermore, they determined one fungi-contributed aerobic respiration pathway could mediate the association between the compromised intestinal barrier and aging.

bioinformatics↗

bioRxiv2022

CspZ FH-binding sites as epitopes promote antibody-mediated Lyme borreliae clearance

Transmitted by ticks, the bacterium Borrelia burgdorferi sensu lato (Bbsl) is the causative agent of Lyme disease (LD), the most common vector-borne disease in the Northern hemisphere. No effective vaccines are currently available. Bbsl produces the CspZ protein that binds to the complement inhibitor, Factor H (FH), promoting evasion of the host complement system. We previously showed that while vaccination with CspZ did not protect mice from Bb infection, mice can be protected after immunization with CspZ-Y207A/Y211A (CspZ-YA), a CspZ mutant protein without FH-binding activity. To further study the mechanism of this protection, herein we evaluated both poly-and monoclonal antibodies recognizing CspZ FH-binding or non-FH-binding sites. We found that the anti-CspZ antibodies that recognize the FH-binding sites (i.e., block FH-binding activity) more efficiently eliminate Bbsl in vitro than those that bind to the non-FH-binding sites, and passive inoculation with anti-FH binding site antibodies eradicated Bbsl in vivo. Antibodies against non-FH-binding sites did not have the same effect. These results emphasize the importance of CspZ FH-binding sites in triggering a protective antibody response against Bbsl in future LD vaccines.

immunology↗

bioRxiv2022

A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model

The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery, which improve vaccination compliance and demonstrate efficacy against emerging variants. Here we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprised of stabilized, pre-fusion Spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction of lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.

microbiology↗

bioRxiv2021

Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. MethodThe 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 and efficacy were evaluated in mice. Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 {+/-} 3.0 mg/L of protein in the 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 as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.

molecular biology↗

bioRxiv2021

Receptor-binding domain recombinant protein RBD219-N1C1 on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

molecular biology↗

bioRxiv2021

A spike-ferritin nanoparticle vaccine induces robust innate immune activity and drives polyfunctional SARS-CoV-2-specific T cells

Potent cellular responses to viral infections are pivotal for long -lived protection. Evidence is growing that these responses are critical in SARS -CoV-2 immunity. Assessment of a SARS -CoV-2 spike ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel(R) (AH) or Army Liposome Formulation containing QS-21 (ALFQ) demonstrated unique vaccine evoked immune signatures. SpFN+ALFQ enhanced recruitment of highly activated classical and non -classical antigen presenting cells (APCs) to the vaccine-draining lymph nodes of mice. The multifaceted APC response of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike -specific T cells with a bias towards TH1 responses and more robust SARS-CoV-2 spike-specific recall response. In addition, SpFN+ALFQ induced Kb spike(539-546)-specific memory CD8+ T cells with effective cytolytic function and distribution to the lungs. This epitope is also present in SARS-CoV, thus suggesting that generation of cross-reactive T cells may provide protection against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant, generates effective SARS-CoV-2 specific innate and adaptive immune T cell responses that are key components to inducing long-lived immunity. One Sentence SummarySpFN vaccine generates multifactorial cellular immune responses.

immunology↗

bioRxiv2021

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques

Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 {micro}g RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only [~]2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. Significance StatementThe emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.

immunology↗

bioRxiv2021

Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 {micro}g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFNs potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses. One-Sentence SummaryA SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

immunology↗

bioRxiv2020

Process Development and Scale-up Optimization of the SARS-CoV-2 Receptor Binding Domain-Based Vaccine Candidate, RBD219-N1C1

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel(R) has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMP) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1 and 5 L scale in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.

immunology↗

bioRxiv2020

Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

BackgroundCoronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. MethodWe constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusionsThese three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significanceBy genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

immunology↗

bioRxiv2020

SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-cell Immunity in Mice

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel(R) were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-{gamma}, IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel(R) containing formulation and possibly in combination with other immunostimulants.

immunology↗

bioRxiv2020

Single-molecule light-sheet microscopy with local nanopipette delivery

Detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2{micro}m thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-{beta}, which causes proteotoxicity relevant to Alzheimers disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells. Statement of SignificanceThis paper describes and validates a new method to study biological processes based on the controlled local delivery of molecules onto or into the cell, combined with single molecule imaging using light sheet microscopy. we not only demonstrate the instruments capability of delivering controlled numbers of molecules to a defined position, down to the level of single molecules, but also its potential in study of the triggering of the innate immune response by protein aggregates, a key process in the development of neurodegenerative diseases such as Alzheimers disease. The same approach could be applied to a wide range of other important biological processes allowing them to be followed in live cells in real-time, hence it will be of great interest to the biophysical community.

biophysics↗