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Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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The ongoing SARS-CoV-2 pandemic continues to sicken millions worldwide and fundamentally change the way people interact with each other. In order to better characterize the SARS-CoV-2 virus and potentially develop methods of inhibition for further spread of the disease, this research project focused on synthesizing and characterizing the trans-membrane region of the accessory protein ORF7a. ORF7a has been implicated in proper viral assembly, leading to the idea that inhibition of this protein could prevent viral copies from being produced and halt the spread of the virus. The goal of this project was to determine the oligomerization state of the protein through a fluorescence assay in order to better understand the quaternary structure of the ORF7a complex and how it folds. The fluorescence assay is performed using three different samples of the synthesized peptide: one labeled with a TAMRA fluorophore, one labeled with a NBD fluorophore, and the last is unlabeled. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Further applications of these inhibitors on other viruses can be explored due to the highly conserved nature of transmembrane domains across multiple viral families. Synthesis of the protein was done using a Solid Phase Peptide Synthesis (SPPS) technique and multiple batches of all three samples of peptide have been generated. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Current research focuses on the purification and quantification of purified ORF7a oligopeptide for implementation of the fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.
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Objectives: Few studies evaluate the immunogenicity and safety of different COVID-19 vaccine platforms in patients with primary Sjogren's Syndrome (pSS). The present study aims to assess the immunogenicity through anti-spike IgG antibodies after the COVID-19 vaccine dose in heterologous groups compared to homologous regimen in patients with pSS. Method(s): These data are from the SAFER study: 'Safety and efficacy of the COVID-19 vaccine in rheumatic disease', a real-life phase IV multicenter longitudinal study, evaluating patients since before the first dose. Pregnant women, those with a history of serious adverse events prior to any vaccine, and those with other causes of immunosuppression were excluded. Patients with pSS > 18 years, classified according to ACR/EULAR 2016 classification criteria were included. Antibodies against the Receptor Binding Domain - RBD portion of the Spike protein of SARS-CoV-2 (IgG-S) were measured by chemiluminescence (Architect SARS-CoV-2 Quanti II, Abbott), before the first dose and 28 days after the 2nd and 3rd dose. Seropositivity was defined as IgG-Spike titers >=7.1 BAU/mL. Patients received adenoviral vector (ChAdOx1, Astrazeneca), mRNA (Pfizer) or inactivated SARS-COV-2 (Coronavac). Non-parametric methods were used. The alpha level of significance was set at 5%. Result(s): 56 participants received 3 doses, 46 +/- 11 years old, disease duration 7.62 years, 92.9% female, 41.1% White and 55.4% Mixed. The homologous third-booster dose group (n = 15, all ChAdOx1) and heterologous group (n = 41) were homogeneous for age, sex, ethnicity, comorbidities, medication and baseline IgG-S median [IQR] titers. After primary vaccination (2 doses) IgG-S median and titers [IQR] were similar in homologous and heterologous groups (373.03 [179.58, 843.92] vs. 473.36 [119.05, 1059.60], p = 0.705). Third-booster dose induced higher IgG-S median [IQR] titers compared to only 2 doses (1229.54 [333.55, 4365.47] vs 464.95 [140.42, 1015.25], p alpha 0.001). Heterologous 3rd-booster induced higher IgG-S median [IQR] titers than homologous scheme with ChAdOx1 (1779.52 [335.83, 4523.89] vs 730.76 [303.37, 1858.98], p = 0.150), Fig 1 and 2, although not statistically significant. Conclusion(s): Third booster dose induced higher humoral immune response compared to two doses whichmay improve protection against COVID-19 in patients with pSS. Although not statistically significant, the response to the heterologous scheme tended to be better than the response to the homologous booster vaccination, which heterologous booster scheme tended to respond better than homologous booster vaccination, which is relevant in this immunosuppressed population. Increasing the sample size will help clarify this issue. .
ABSTRACT
Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.
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Objectives: To evaluate the humoral immune response to the third dose (booster) of vaccine against SARS-CoV-2 in patients with autoimmune rheumatic diseases who were seronegative after a two-dose regimen. Method(s): Observational study. Patients with autoimmune rheumatic diseases who had not achieved seroconversion after a two-dose vaccine schedule against SARS-CoV-2 were included. To assess the humoral immune response, anti-RBD IgG (S protein receptor binding domain) neutralizing antibody titers were determined by ELISA (cutoff titer 200). The determination was made between 30 to 45 days after the third dose. Result(s): From 66 patients who received SARS-CoV-2 vaccination, 18 patients (29.5%) were seronegative after a two-dose schedule. 61% had SLE, 77% had comorbidities (61% with hypertension, p = 0.03). Patients were on treatment: 10 with prednisone (8 with doses greater than 10 mg/d, p = 0.01), 10 with hydroxychloroquine, one with methotrexate, one with leflunomide, four with azathioprine, five with my cophenolatemofetil and five with rituximab (they are the total number of non-responders on biological treatment, p = 0.03). Regarding the primary vaccination regimen, 11 received BBIBP-CorV (p = 0.01), 5 AZD1222, 1 Gam-COVID-Vac and 1 mRNA1273/Gam-COVID-Vac heterologous scheme. Of these 18 non-responders, 14 received a third dose;nine patients (62%) presented anti-RBD IgG detectable. Of the five patients who did not respond to the booster vaccination, three had received BBIBP-CorV as the initial schedule and the vaccines applied as a third dose were Ad5-nCoV (1), BNT162b2 (1), AZD 1222 (2) and Gam-COVID-Vac (1). They were being treated with: rituximab (2), azathioprine (2) and mycophenolate mofetil (1). Treatment with higher doses of prednisone was the only factor associated with non-seroconversion to the third dose (8 +/- 4.5;p 0.02). Conclusion(s): The third dose of SARS-CoV-2 vaccine allowed to improve the serological response to vaccination, achieving a seroconversion of 62% in this group of patients.
ABSTRACT
Objectives: Patients with immune-mediated rheumatic diseases (IMRDs) develop more severe outcomes of Coronavirus disease 2019 (COVID-19). Recent studies have contributed to understand the safety and efficacy of COVID-19 vaccines in IMRDs, suggesting that different diseases and therapies may interfere on immunization efficacy. In this study we analyze the immunogenicity of COVID-19 vaccines in patients with Systemic Vasculitides (VASC), the rate of COVID-19 and the frequency of disease relapse following immunization. Method(s): We included patients with VASC (n = 73), a subgroup of the SAFER study (Safety and Efficacy on COVID-19 Vaccine in Rheumatic Disease), a longitudinal, multicenter, Brazilian cohort.We analyzed the geometric means of IgG antibody against receptor-biding domain of protein spike of SARS-CoV-2 (anti-RBD) after two shots of CoronaVac (Inactivated vaccine), ChadOx-1 (AstraZeneca) or BNT162b2 (Pfizer-BioNTech). IgG anti-RBD was measured by chemiluminescence test. We assessed new-onset COVID-19 episodes, adverse events (AE) and disease activity for each VASC. Result(s): The sample included Behcet's disease (BD) (n = 41), Takayasu arteritis (TAK) (n = 15), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) (n = 14), polyarteritis nodosa (n = 7) and other small vessel VASC(n = 6). The majority of patients were female (69%) without comorbidities (49%) and a median age of 37 years. The most common medication was conventional synthetic disease-modifying anti-rheumatic drugs, followed by biologic drugs. No patient received rituximab at baseline. Most patients received CoronaVac (n = 25) or ChadOx-1 (n = 36), while four received BNT162b2. Baseline IgG-RBD means were 1.34 BAU/mL. They increased to 3.89 and 5.29 BAU/mL after the 1st and 2nd vaccine dose, respectively. ChadOx-1 had higher antibody titers than CoronaVac (p = 0.002). There were no differences between different VASC. There were 3 cases of COVID-19 after immunization with CoronaVac. BD patients had a tendency for more cutaneous-articular activity following ChadOx-1. There were no severe relapses and no serious adverse events. Conclusion(s): Our results show the safety of different SARS-CoV-2 vaccines in VASC population. A progressive increase of IgG-RBD antibodies was observed after each dose. ChadOx-1 led to higher IgG-RBDgeometricmeans compared toCoronaVac. Finally, even though ChadOx-1 presented a tendency of triggering mild disease activity, there were no significant disease activity following vaccination in VASC patients. .
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Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023
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Background: More than 12 billion doses of COVID-19 vaccine administrations and over 630 million natural infections should have developed adequate levels of herd immunity over the last three years. However, there have been many new waves of coronavirus infections. The development of safe and effective vaccines to control breakthrough SARS-CoV-2 infections remain an urgent priority. We have developed a recombinant VSV vector-based vaccine to fulfill this worldwide need. Method(s): We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 recall vaccine candidate. We have constructed an attenuated recombinant VSV genome carrying the full-length Spike protein gene of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) at the N-terminus enhanced the protein expression and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) at the C-terminus of the Spike protein allowed efficient incorporation of the Spike protein into pseudotype VSV. Result(s): In immunized mice, rVSV with chimeric rVSV-msp-S-Gtc induced high levels of potent neutralizing antibodies (nAbs) and CD8+ T cell responses, while the full-length Spike with Gtc proved to be the superior immunogen. More importantly, rVSV-msp-S-Gtc-vaccinated animals were completely protected from subsequent SARS-CoV-2 challenges. Furthermore, rVSV-Wuhan and rVSV-Delta vaccines, and an rVSV-Trivalent (mixed rVSV-Wuhan, -Beta and -Delta) vaccine elicited potent nAbs against live SARS-CoV-2 Wuhan (USAWA1), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) viruses. Heterologous boosting of rVSV-Wuhan with rVSV-Delta induced strong nAb responses against Delta and Omicron viruses, with the rVSV-Trivalent vaccine consistently inducing effective nAbs against all the SARS-CoV-2 variants tested. All rVSV-msp-S-Gtc vaccines also elicited an immunodominant Spike-specific CD8+ T cell response. Conclusion(s): rVSV vaccines targeting SARS-CoV-2 variants of concern can be considered as an effective booster vaccine in the global fight against COVID-19.
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Introduction/Aim: The development of safe and effective vaccines is crucial to conquering the COVID-19 pandemic. Recombinant proteins represent the best understood and reliable approach to pandemic vaccine delivery with well-established safety;however, they face challenges in design, structural characterisation, manufacture, potency testing and ensuring adequate immunogenicity. Method(s): Our team used in silico structural modelling to design a vaccine based on a stabilised spike protein extracellular domain (ECD). The insect cell expressed recombinant spike ECD was formulated with Vaxine's proprietary Advax-CpG55.2 adjuvant. Result(s): The vaccine known as Covax-19 or SpikoGen induced high titers of antibody and memory T-cells which translated to protection against SARS-CoV-2 infection in hamsters, ferrets, and aged monkeys. Despite numerous challenges along the journey, clinical trials in Iran during a major wave of delta variant infection confirmed SpikoGen vaccine was 78% effective in reducing risk of severe disease and with no evidence of vaccine-associated thrombosis, myocarditis, or sudden death, receiving marketing approval under emergency use authorisation in Iran on 6 October 2021. This made it the first recombinant spike-protein vaccine in the world to be approved, and the first Australian-developed human vaccine to receive marketing approval in four decades. Since approval millions of doses have been administered and additional trials in Australia and Iran have confirmed its effectiveness as a booster to prevent waning immunity, as well as its safety and effectiveness in children from the age of 5 years. The ongoing Australian and overseas clinical trial program is focussed on gaining better understanding the effect of dosing intervals on vaccine immunogenicity, gathering additional data on use as a booster, and development of new variant formulations. Conclusion(s): Covax-19/Spikogen is safe and effective adjuvanted recombinant protein vaccine.
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The transmembrane domains of viral proteins are highly conserved and crucial to normal viral function. Oligomeric transmembrane domains present novel opportunities for drug development, as their disruption can prevent the assembly of the virus. The Reichart lab is particularly interested in developing retro-inverso peptide inhibitors. Retro-inverso peptides are peptides using D-amino acids mirroring a region of target protein, which allows the peptide to inhibit viral assembly, but they are also significantly less likely to be catabolized by natural metabolic or immunologic processes. The efficacy of these inhibitors is governed largely by the extent to which they mirror the target protein, making highly conserved regions, such as transmembrane domains, ideal target regions for these inhibitors. The primary technique in the literature for the investigation of oligomerization states uses fluorescence spectroscopy. We are now working on developing a novel alternative system to evaluate protein oligomerization using spin-labeled peptides that are directly incorporated into the peptide sequence. Direct incorporation of the spin-label into the peptide sequence is a more powerful technique than the standard procedures used in the literature. In particular, the ability to incorporate spin labels in various positions within the protein can give novel insights into the relative depth of the protein within a membrane, which is very difficult to study using other techniques and not possible using the fluorescence technique. The transmembrane domains of proteins with known and well-characterized monomer and trimer standard oligomerization states were synthesized using an Fmoc Solid- Phase Peptide Synthesis (SPPS) procedure incorporating an Fmoc-2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, (Fmoc-TOAC) instead of an alanine. Direct incorporation of stable N-oxide spin labels, which can be contrasted to labeling cysteine residues after the protein synthesis, has been used for the investigation of the secondary structure of proteins for decades, but the application of this spin labeling technique to study the oligomerization states of transmembrane domains of proteins is an understudied application. The products of SPPS were analyzed using a Liquid Chromatography Mass Spectroscopy instrument and purified using High Performance Liquid Chromatography. The spin-label was then deprotected and evaluated using Electron Spin Resonance (ESR) Spectroscopy. There are two primary future directions following this research project: first, the generation of viral proteins with spin labels incorporated in different positions to determine the relative depth of each position within the membrane;second, the incorporation of spin labels into SARS-CoV- 2 proteins to develop a model for in vitro evaluation of retro-inverso peptide assembly inhibitors. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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The spike protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is directly responsible for the binding to ACE2 receptors in host cells. While the spike protein overall is known to form trimers, the oligomerization state of the transmembrane domain of the spike protein in SARS-CoV-2 is unknown. It is believed to be essential for the function of this protein. Since the transmembrane domain of the spike protein is highly conserved in SARS-CoV-2 it is important to investigate its character and determine its relationship to the function of the protein as awhole. The goal of this project was to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the spike protein in SARS-CoV-2. The most practical method to synthesize the TM domain of the S protein is through solid phase peptide synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will then need to be purified using high-performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography- mass spectrometry (LCMS) to confirm the identity of the synthesized peptides as well as any potential impurities. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS-CoV-2. The function of the S protein is believed to only present when the transmembrane domain forms a trimer. Therefore, the analysis of their oligomerization states will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and tetramethylrhodamine (TAMRA) in a fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
The presence of estrogenic compounds (endocrine-disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time-consuming techniques such as liquid chromatography-mass spectrometry and high-performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our approach to the design of an estrogen responsive element uses methodology developed in the Wells lab (Elledge et al, 2021) to detect SARS-CoV-2 antibodies. This methodology takes advantage of the split Nanoluciferase (spLUC) protein divided into two functional domains (designated SmBit and LgBit). Based on rational engineering design we express dimerization dependent LgBit and SmBit fused to the Estrogen Receptor alpha protein (ERalpha) in bacteria cells. These two monomeric proteins will dimerize in the presence of estrogen, reconstitute the split luciferase enzyme and reestablish enzyme activity. Cells can be lysed, and luminescence detected to quantify estrogen present in the sample. We present here the construction strategy and proof of concept data demonstrating the efficiency of this dual-functional biosensor and its effectiveness for detection of estrogenic compounds in contaminated water. NSF-REU-1852150, REU Site: A multisite REU in Synthetic Biology, 2019.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.
ABSTRACT
The novel COVID-19 vaccines have been instrumental at transforming the pandemic into an endemic disease. However, many contemporary vaccines, especially the landmark mRNA vaccines, require cold storage that makes them difficult for low income and developing countries to keep and distribute, and no shelf stable, low-cost alternative currently exists. In response to this need, we are developing a novel COVID-19 vaccine delivery system using the probiotic yeast Saccharomyces boulardii. We engineered an integrating construct to express the receptor binding domain (RBD) of the SARS-CoV-2 spike protein tagged with the yeast pheromone secretion signal and with the Claudin-4 targeting sequence of the Clostridium perfringens enterotoxin. Preliminary data from two animal trials suggest that our candidate yeast oral COVID-19 vaccine can trigger a robust humoral immune response in mice. Experiments are underway to assess its effect on the murine T-cell response. Our laboratory is supported in part by a research grant from the PCHRD-DOST of the Republic of the Philippines.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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Purpose of Study: Antibiotic resistance remains one of the largest healthcare and public health challenges. Several studies have documented that the spread of antibiotic resistant bacteria in nosocomial settings has been exacerbated worldwide due to increased rates of hospitalization and intubation in the wake of the COVID-19 pandemic. One way to address antibiotic resistance is to identify novel compounds that inhibit essential microbial processes. Two-component regulatory systems are important mediators of signal transduction that allow bacteria to communicate with and respond to changes in their environment. The WalRK system is a two-component system that is conserved and essential for viability in many Gram-positive human pathogens. We hypothesize that a ligand that specifically binds with the DNA-interaction surface of the WalR protein can lead to cell death and can serve as a lead compound for future drug development efforts. Methods Used: We describe the development process of an assay to identify WalR binding compounds. In silico molecular dynamics docking approaches were utilized to identify potential WalR binding compounds from virtual compound libraries. To assess their WalR-binding capacity in vitro, overexpression strains for several WalR recombinant constructs were engineered and protein constructs were purified to homogenicity. Isothermal titration calorimetry (ITC) is a technique that measures heat release or absorption when two molecules interact. A MicroCal PEAQ ITC instrument was utilized to develop a WalR-binding assay. Summary of Results: WalR is a two-domain protein featuring a regulatory and a DNA-binding domain. Two constructs, a truncated DNA-binding domain and a full-length protein construct proved soluble, and pure quantities necessary to conduct ITC measurements could be successfully obtained (12 mg full-length protein and 23 mg truncated protein). These proteins were amenable to ITC experiments. We found that experiments were best run with at least a two-fold increase of ligand concentration to protein concentration supplied in identical buffer conditions over nineteen injections. We are currently assessing the binding affinities of our in silico hit compounds. Conclusion(s): Our results show that ITC enables the detailed, rapid, and reproducible characterization of the binding relationship between the DNA-binding domain of the WalR protein and any potential ligands. The protocol discussed herein will enable further drug discovery studies on the WalR response regulator protein to identify and characterize inhibitors, providing insight towards the development of novel antimicrobial compound.
ABSTRACT
Background. Vaccine-induced SARS-CoV-2 antibody responses are reduced in patients taking lymphocyte-depleting therapies, which are commonly prescribed for patients with idiopathic inflammatory myopathies (IIM). While a third vaccine dose (D3) augments the SARS-CoV-2 anti-spike response in some patients, there is a paucity of data on the humoral response following D3 in patients with IIM. Furthermore, the durability of antibody response is unknown. In this study, we evaluated serial antibody response for three months following a 3rd dose SARS-CoV-2 vaccination in IIM patients. Methods. Adults with a patient-reported diagnosis of idiopathic inflammatory myopathy who completed three-dose SARS-CoV-2 vaccination (two-dose BNT162b2 or mRNA-1273 followed by single mRNA or adenoviral vector dose) were recruited via social media campaign. Demographics and clinical characteristics were collected via patient report. Informed consent was provided electronically. Serial antibody responses were evaluated by the Roche Elecsys anti-SARS-CoV-2 S enzyme immunoassay, which measures total antibody to the SARS-CoV-2 S-receptor binding domain (RBD) protein (range 0. 4-2500U/ mL;positive >0.8U/mL). Poor antibody response was defined as anti-RBD titer <500U/mL based on predicted correlates of protective plasma neutralizing capacity. Those with prior COVID-19 infection were excluded. Associations were evaluated using Fisher's exact and Wilcoxon rank-sum tests as appropriate. Results. We evaluated serial anti-RBD titers in 59 participants (Table I). Most (93%) were female with median (IQR) age of 51 (41-62) years. Mycophenolate mofetil was the most frequently prescribed medication (45.6%). Participants completed primary vaccination with two-dose BNT162b2(54%) or mRNA-1273(46%). Median pre-D3 anti-RBD titer (IQR) was 65.8U/mL (4.6,473) at 158 (136-183) days following primary vaccination. Dose 3 included BNT162b2(47%), mRNA-1273(47%) or Ad.26.COV2.S (6%). Most (89.9%) received homologous D3 vaccination. 39% of participants reported holding peri-D3 immunosuppression with mycophenolate mofetil being the most commonly held medication in the peri-D3 period. Repeat anti-RBD testing was performed at a median (IQR) 30 (28-32) days post-D3. A higher antibody titer was seen in 89.9% participants following D3 with median (IQR) titer of 2500 U/mL (92,2500). Thirty-seven percent remained <500U/mL following D3;a greater proportion of these participants reported use of rituximab and greater number of immunosuppressive therapies compared to those with anti-RBD >=500U (72.7% versus 5.4%, p<0.001;3 therapies versus 2 therapies, p=0.03). Furthermore, 13.5% (8/59) remained below the threshold of positivity following D3;7/8 reported use of rituximab, 5/8 mycophenolate mofetil, or combination of these agents (4/8). There was not a significant difference in antibody titers among recipients of homologous/heterologous vaccination (p=0.22). Dose 3 was well tolerated with only 2 (3.4%) participants reporting disease flare requiring treatment within one month of vaccination;neither required intravenous therapy or hospital admission. Thirty-four (57.6%) participants underwent repeat anti-RBD testing three months following D3 with median (IQR) 2500U/mL (456,2500);73.53% (25/34) remained above threshold of >=500U/mL. Limitations of this study include small sample size and absence of healthy control group. Diagnosis was based on participant report and we did not routinely collect information on disease activity. Conclusion. We observed an augmented humoral response in most IIM patients following 3rd dose SARS-CoV-2 vaccination;antibody response was durable at three months. Dose 3 was well tolerated. Over 1/3 participants failed to develop adequate response following D3, namely those on rituximab therapy and on higher number of immunosuppressive therapies. These patients should be prioritized for prophylactic therapies to enhance protection against COVID-19 infection.
ABSTRACT
Asymptomatic subjects account for 25 to 45% of SARS-CoV-2 infections, and in particular, subjects on mild immunosuppressive therapy may have symptoms masked and could spread virus for an extended period of time. To determine the cumulative incidence of symptomatic and asymptomatic SARS-CoV-2 infections and associated risk factors, we conducted a prospective clinical and serological survey in a cohort of 278 liver transplant recipients (LTRs) from Central Italy. Three different serology tests were performed every 4 months in 259 LTRs between April 2020 and April 2021: one based on raw extract of whole SARS-CoV-2 virus and two on specific viral antigens (nucleoprotein and receptor binding domain) to detect specific IgG, IgM and IgA. Hundred fifteen LTRs who reported symptoms or close contact with a SARS-CoV-2-positive subject, or had a positive serological result underwent molecular testing by standard screening procedures (RT-PCR on naso-pharyngeal swab). Thirty-one past or active SARS-CoV-2 infections were identified: 14 had positive molecular test (64% symptomatic), and 17 had positive serology only (18% symptomatic). SARS-CoV-2 infection was not statistically related to gender, age, obesity, diabetes, renal impairment, type of anti-rejection therapy or time from transplant. Asymptomatic SARS-CoV-2 cases (61.3%) were more frequent in males and in those with glomerular filtrate rate >50 ml/min. Overall, the addition of repeated serology to standard diagnostic molecular protocols increased detection of SARS-CoV-2 infection from 5.1% to 10.9%. Anti-SARS-CoV-2 seroprevalence among our LTRs (11.2%) is comparable to the general population of Central Italy, considered a medium-impact area. Only one asymptomatic subject (6%) was found to carry SARS-CoV-2 in respiratory tract at the time of serological diagnosis.Copyright © 2021 The Authors
ABSTRACT
Antimicrobial photodynamic therapy (aPDT) [1] has been deployed in tens of thousands of patients in Canada for preoperative intranasal bacterial suppression to reduce the prevalence rate of surgical site infections [2]. This treatment has proven safe and effective, with infection rate reductions of 40-80% in tertiary care systems despite only requiring 4 minutes of therapy [2]. We previously demonstrated that aPDT eliminates the RNA signature of wild-type SARS-CoV-2 in vitro, with reduction of RT-qPCR threshold counts (DELTACt = 22) in a light-dose dependent manner (C = 320 muM, lambda = 664 nm, F = 36 J/cm2) [3]. Photodynamic targets were found to include the receptor binding domain, spike protein and nucleocapsid domain, consistent with a broad spectrum peroxidative effect on anionic moieties throughout the virion [3]. This work describes the benefits of using regular aPDT treatments in the industrial workplace for the purpose of employee COVID-19 prevention. From July 2020 to August 2021, aPDT was deployed at a large Canadian food processing plant. Meat processing facilities face distinctive challenges in control of infectious diseases, including SARS-CoV-2. Factors that increase processing workers' risk for exposure to SARS-CoV-2 include close contact for 8-12 hour shifts, shared transportation, and congregate housing [4,5]. The presence of a slaughtering plant in a community is associated with a 51 to 75% increase in COVID-19 cases per thousand over the baseline community rate, and a 37 to 50% increase in death rate over the baseline community rate [5]. Methylene blue-mediated aPDT (SteriwaveTM Nasal Photodisinfection System, Ondine Biomedical Inc., Vancouver, BC) was added to the standard infection control bundle at the plant, along with employee education. Treatments were administered free of charge to approximately 1,500 employees on a voluntary basis during paid work hours. Compliance levels of employees requesting aPDT were 85%. To determine intervention efficacy, the rate of qPCR-positive COVID-19 tests over the treatment time period was compared to the same rate in the surrounding province. Results demonstrated a reduction of COVID-19 rate of over 3 times (p<.0001, Fisher's Exact Test) in the treated population compared to the untreated population, with the largest adverse event being mild (self-limiting) rhinorrhea in < 1% of cases. The plant continued production and distribution of products without disruption. Important outcomes from this quality improvement initiative included (a) aPDT proved to be a rapid, lightweight intervention that could be deployed at high compliance levels in a commercial high-throughput food processing operation, (b) significant impact (>3X reduction) on the COVID-19 rates was observed and (c) COVID-19-related comorbidities including acute and long-term illness, disability, and death were proportionately avoided.Copyright © 2023
ABSTRACT
The severe consequences of the Zika virus (ZIKV) infections resulting in congenital Zika syndrome in infants and the autoimmune Guillain-Barre syndrome in adults warrant the development of safe and efficacious vaccines and therapeutics. Currently, there are no approved treatment options for ZIKV infection. Herein, we describe the development of a bacterial ferritin-based nanoparticle vaccine candidate for ZIKV. The viral envelope (E) protein domain III (DIII) was fused in-frame at the amino-terminus of ferritin. The resulting nanoparticle displaying the DIII was examined for its ability to induce immune responses and protect vaccinated animals upon lethal virus challenge. Our results show that immunization of mice with a single dose of the nanoparticle vaccine candidate (zDIII-F) resulted in the robust induction of neutralizing antibody responses that protected the animals from the lethal ZIKV challenge. The antibodies neutralized infectivity of other ZIKV lineages indicating that the zDIII-F can confer heterologous protection. The vaccine candidate also induced a significantly higher frequency of interferon (IFN)-γ positive CD4 T cells and CD8 T cells suggesting that both humoral and cell-mediated immune responses were induced by the vaccine candidate. Although our studies showed that a soluble DIII vaccine candidate could also induce humoral and cell-mediated immunity and protect from lethal ZIKV challenge, the immune responses and protection conferred by the nanoparticle vaccine candidate were superior. Further, passive transfer of neutralizing antibodies from the vaccinated animals to naïve animals protected against lethal ZIKV challenge. Since previous studies have shown that antibodies directed at the DIII region of the E protein do not to induce antibody-dependent enhancement (ADE) of ZIKV or other related flavivirus infections, our studies support the use of the zDIII-F nanoparticle vaccine candidate for safe and enhanced immunological responses against ZIKV.