TY - JOUR TI - Toward the quantification of adeno-associated virus titer by electrochemical impedance spectroscopy AU - Wang, Junhyeong AU - Hosseini, Mahshid AU - Shastry, Shriarjun AU - Barbieri, Eduardo AU - Chu, Wenning AU - Menegatti, Stefano AU - Daniele, Michael A. T2 - 2023 IEEE BIOSENSORS CONFERENCE, BIOSENSORS AB - Gene therapies have shown great promise for the potential treatment of a broad range of diseases. Adeno-associated viruses (AAVs) are popular gene vectors because of their ability to target specific tissues, and they have demonstrated high transduction efficiencies in multiple neurological targets. While these therapeutics hold great promise, their biomanufacturing has limited potential cost-reduction and more widespread adoption. Herein, we report the preliminary development of an immunosensor for measuring the titer of adeno-associated virus 2 (AAV2), which may be deployed for rapid quantification of product yield during AAV biomanufacturing. We functionalized an interdigitated electrode array with anti-AAV2 antibodies, and electrochemical impedance spectroscopy was employed to investigate the response to AAV2 titer. A Faradaic sensing principle was utilized, in which the charge transfer resistance (Rct) of an electrochemical reporter was monitored after capture of AAV2 on the surface of the sensor. A linear response was measured over titers 1012 - 1013 capsids/mL. DA - 2023/// PY - 2023/// DO - 10.1109/BioSensors58001.2023.10281105 SP - KW - adeno-associated virus KW - AAV2 KW - immunosensor KW - electrochemical impedance spectroscopy KW - viral vector ER - TY - JOUR TI - The downstream bioprocess toolbox for therapeutic viral vectors AU - Kilgore, Ryan AU - Minzoni, Arianna AU - Shastry, Shriarjun AU - Smith, Will AU - Barbieri, Eduardo AU - Wu, Yuxuan AU - Lebarre, Jacob P. AU - Chu, Wenning AU - O'Brien, Juliana AU - Menegatti, Stefano T2 - JOURNAL OF CHROMATOGRAPHY A AB - Viral vectors are poised to acquire a prominent position in modern medicine and biotechnology owing to their role as delivery agents for gene therapies, oncolytic agents, vaccine platforms, and a gateway to engineer cell therapies as well as plants and animals for sustainable agriculture. The success of viral vectors will critically depend on the availability of flexible and affordable biomanufacturing strategies that can meet the growing demand by clinics and biotech companies worldwide. In this context, a key role will be played by downstream process technology: while initially adapted from protein purification media, the purification toolbox for viral vectors is currently undergoing a rapid expansion to fit the unique biomolecular characteristics of these products. Innovation efforts are articulated on two fronts, namely (i) the discovery of affinity ligands that target adeno-associated virus, lentivirus, adenovirus, etc.; (ii) the development of adsorbents with innovative morphologies, such as membranes and 3D printed monoliths, that fit the size of viral vectors. Complementing these efforts are the design of novel process layouts that capitalize on novel ligands and adsorbents to ensure high yield and purity of the product while safeguarding its therapeutic efficacy and safety; and a growing panel of analytical methods that monitor the complex array of critical quality attributes of viral vectors and correlate them to the purification strategies. To help explore this complex and evolving environment, this study presents a comprehensive overview of the downstream bioprocess toolbox for viral vectors established in the last decade, and discusses present efforts and future directions contributing to the success of this promising class of biological medicines. DA - 2023/10/25/ PY - 2023/10/25/ DO - 10.1016/j.chroma.2023.464337 VL - 1709 SP - SN - 1873-3778 KW - Viral vectors KW - Gene therapy KW - Affinity ligands KW - Chromatography KW - Adsorbents KW - AAV ER - TY - JOUR TI - Pseudo-affinity capture of K. phaffii host cell proteins in flow-through mode: Purification of protein therapeutics and proteomic study AU - Sripada, Sobhana A. AU - Elhanafi, Driss AU - Collins, Leonard B. AU - Williams, Taufika I. AU - Linova, Marina Y. AU - Woodley, John M. AU - Boi, Cristiana AU - Menegatti, Stefano T2 - SEPARATION AND PURIFICATION TECHNOLOGY AB - K. phaffii is a versatile expression system that is increasingly utilized to produce biological therapeutics – including enzymes, engineered antibodies, and gene-editing tools – that feature multiple subunits and complex post-translational modifications. Two major roadblocks limit the adoption of K. phaffii in industrial biomanufacturing: its proteome, while known, has not been linked to downstream process operations and detailed knowledge is missing on problematic host cell proteins (HCPs) that endanger patient safety or product stability. Furthermore, the purification toolbox has not evolved beyond the capture of monospecific antibodies, and few solutions are available for engineered antibody fragments and other protein therapeutics. To unlock the potential of yeast-based biopharmaceutical manufacturing, this study presents the development and performance validation of a novel adsorbent – PichiaGuard – functionalized with peptide ligands that target the whole spectrum of K. phaffii HCPs and designed for protein purification in flow-through mode. The PichiaGuard adsorbent features high HCP binding capacity (∼25 g per liter of resin) and successfully purified a monoclonal antibody and an ScFv fragment from clarified K. phaffii harvests, affording > 300-fold removal of HCPs and high product yields (70–80%). Notably, PichiaGuard outperformed commercial ion exchange and mixed-mode resins without salt gradients or optimization in removing high-risk HCPs – including aspartic proteases, ribosomal subunits, and other peptidases – thus demonstrating its value in modern biopharmaceutical processing. DA - 2023/12/1/ PY - 2023/12/1/ DO - 10.1016/j.seppur.2023.124777 VL - 326 SP - SN - 1873-3794 KW - Komagataella phaffii KW - Chromatography KW - Protein purification KW - Monoclonal antibodies KW - Antibody fragment KW - Proteomics ER - TY - JOUR TI - Draft genome sequences of a historical collection of Listeria monocytogenes from humans and other sources, 1926-1964 AU - Brown, Phillip AU - Murray, Robert G. E. AU - Galsworthy, Sara AU - Ivanova, Mirena AU - Leekitcharoenphon, Pimlapas AU - Ward, Todd AU - Kucerova, Zuzana AU - Chen, Yi AU - Elhanafi, Driss AU - Siletzky, Robin AU - Kathariou, Sophia T2 - MICROBIOLOGY RESOURCE ANNOUNCEMENTS AB - Listeria monocytogenes can persistently contaminate food processing environments and tolerate sanitizers. Most sequenced strains are from clinical and environmental sources in the contemporary era, with relatively few prior to extensive food processing and sanitizer use. We report the genome sequences of a diverse panel of 83 strains from 1926 to 1964. DA - 2023/9/29/ PY - 2023/9/29/ DO - 10.1128/MRA.00625-23 SP - SN - 2576-098X KW - Listeria monocytogenes KW - food-borne pathogens KW - listeriosis KW - DNA sequencing KW - genomes KW - intracellular pathogens KW - historical strains ER - TY - JOUR TI - Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography AU - Shastry, Shriarjun AU - Chu, Wenning AU - Barbieri, Eduardo AU - Greback-Clarke, Paul AU - Smith, William K. AU - Cummings, Christopher AU - Minzoni, Arianna AU - Pancorbo, Jennifer AU - Gilleskie, Gary AU - Ritola, Kimberly AU - Daniele, Michael A. AU - Johnson, Thomas F. AU - Menegatti, Stefano T2 - BIOTECHNOLOGY JOURNAL AB - Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. In particular, the AAV purification pipeline hinges on protein ligands for the affinity-based capture step. While featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product's activity and stability. Additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades - namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (≈1014 vp mL-1 ) and product yields (≈60%-80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%). DA - 2023/9/28/ PY - 2023/9/28/ DO - 10.1002/biot.202300230 VL - 9 SP - SN - 1860-7314 KW - adeno-associated virus KW - affinity chromatography KW - gene therapy KW - peptide ligands KW - transduction activity ER - TY - CHAP TI - Digital Twins in Pilot Scale Fermentation: Non-Linear State Estimation for Improving Induction Timing AU - Stevnsborg, Mads AU - Selle, Kurt AU - Barton, Ryan AU - Prado-Rubio, Oscar A. AU - Gargalo, Carina AU - Gernaey, Krist V. AU - Gilleskie, Gary AU - Huusom, Jakob K. T2 - Computer Aided Chemical Engineering AB - In this work, a model is developed and implemented for GFPUV production with aerobic fed-batch fermentation of E coli BL21 (DE3). The model parameters are estimated using historical process data and minimizing the prediction and measurement error. The model implements an extended Kalman filter for non-linear state estimation of biomass, glucose, and dissolved oxygen concentration. The filter includes an existing cascade feed-back loop for dissolved oxygen control which improves the predictive accuracy of the filter. The estimator is used during fermentation to predict the induction point based on a threshold glucose concentration which is otherwise determined exclusively with at-line measurements. The validation examples presented in this work show great agreement between the estimated and measured glucose concentrations, making it a useful tool for predicting the time until induction without requiring high-frequency at-line sampling. PY - 2023/// DO - 10.1016/b978-0-443-15274-0.50419-4 SP - 2637-2642 PB - Elsevier SN - 9780443152740 UR - http://dx.doi.org/10.1016/B978-0-443-15274-0.50419-4 ER - TY - JOUR TI - Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates AU - Chu, Wenning AU - Shastry, Shriarjun AU - Barbieri, Eduardo AU - Prodromou, Raphael AU - Greback-Clarke, Paul AU - Smith, Will AU - Moore, Brandyn AU - Kilgore, Ryan AU - Cummings, Christopher AU - Pancorbo, Jennifer AU - Gilleskie, Gary AU - Daniele, Michael A. AU - Menegatti, Stefano T2 - BIOTECHNOLOGY AND BIOENGINEERING AB - Abstract Adeno‐associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV‐based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands—typically camelid antibodies—that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH = 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1‐VP2 and VP2‐VP3 virion proteins with mild binding strength ( K D ~ 10 −5 –10 − 6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC 10% > 10 13 vp/mL of resin) and product yields (~50%–80%) on par with commercial adsorbents. The peptide‐based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50%–80%), 80‐ to 400‐fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses. DA - 2023/7/12/ PY - 2023/7/12/ DO - 10.1002/bit.28495 VL - 7 SP - SN - 1097-0290 KW - adeno-associated virus KW - affinity chromatography KW - HEK 293 lysate KW - peptide ligands KW - virus purification ER - TY - JOUR TI - Advances in high-throughput, high-capacity nonwoven membranes for chromatography in downstream processing: A review AU - Lavoie, Joseph AU - Fan, Jinxin AU - Pourdeyhimi, Behnam AU - Boi, Cristiana AU - Carbonell, Ruben G. T2 - BIOTECHNOLOGY AND BIOENGINEERING AB - Nonwoven membranes are highly engineered fibrous materials that can be manufactured on a large scale from a wide range of different polymers, and their surfaces can be modified using a large variety of different chemistries and ligands. The fiber diameters, surface areas, pore sizes, total porosities, and thicknesses of the nonwoven mats can be carefully controlled, providing many opportunities for creative approaches for the development of novel membranes with unique properties to meet the needs of the future of downstream processing. Fibrous membranes are already finding use in ultrafiltration, microfiltration, depth filtration, and, more recently, in membrane chromatography for product capture and impurity removal. This article summarizes the various methods of manufacturing nonwoven fabrics, and the many methods available for the modification of the fiber surfaces. It also reviews recent studies focused on the use of nonwoven fabric devices in membrane chromatography and provides some perspectives on the challenges that need to be overcome to increase binding capacities, decrease residence times, and reduce pressure drops so that eventually they can replace resin column chromatography in downstream process operations. DA - 2023/5/31/ PY - 2023/5/31/ DO - 10.1002/bit.28457 SP - SN - 1097-0290 KW - downstream purification KW - fibrous systems KW - membrane adsorbers KW - membrane chromatography KW - nonwovens KW - product capture and polishing ER - TY - JOUR TI - Draft Genome Sequences of 158 Listeria monocytogenes Strains Isolated from Black Bears (Ursus americanus) in the United States AU - Brown, Phillip AU - Chen, Yi AU - Ivanova, Mirena AU - Leekitcharoenphon, Pimlapas AU - Parsons, Cameron AU - Niedermeyer, Jeffrey AU - Gould, Nicholas AU - Strules, Jennifer AU - Mesa-Cruz, J. Bernardo AU - Kelly, Marcella J. AU - Hooker, Michael J. AU - Chamberlain, Michael J. AU - Olfenbuttel, Colleen AU - DePerno, Christopher AU - Elhanafi, Driss AU - Kathariou, Sophia T2 - MICROBIOLOGY RESOURCE ANNOUNCEMENTS AB - Listeria monocytogenes is responsible for severe foodborne disease and major economic losses, but its potential reservoirs in natural ecosystems remain poorly understood. Here, we report the draft genome sequences of 158 L. monocytogenes strains isolated from black bears ( Ursus americanus ) in the southeastern United States between 2014 and 2017. DA - 2023/6/5/ PY - 2023/6/5/ DO - 10.1128/mra.00248-23 SP - SN - 2576-098X ER - TY - JOUR TI - Draft Genome Sequences of Closely Related Listeria monocytogenes Lineage III Strains Isolated from a Food Processing Environment and a Case of Human Listeriosis AU - Brown, Phillip AU - Kanenaka, Rebecca AU - Chen, Yi AU - Ivanova, Mirena AU - Leekitcharoenphon, Pimlapas AU - Elhanafi, Driss AU - Kathariou, Sophia T2 - MICROBIOLOGY RESOURCE ANNOUNCEMENTS AB - Listeria monocytogenes lineage III is genetically highly diverse, and closely related lineage III strains from food facilities and human listeriosis have not been reported. Here, we report the genome sequences of three closely related lineage III strains from Hawaii, namely, one isolated from a human case and two isolated from a produce storage facility. DA - 2023/5/22/ PY - 2023/5/22/ DO - 10.1128/mra.00250-23 SP - SN - 2576-098X ER - TY - JOUR TI - Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane AU - Fan, Jinxin AU - Sripada, Sobhana A. AU - Pham, Dan N. AU - Linova, Marina Y. AU - Woodley, John M. AU - Menegatti, Stefano AU - Boi, Cristiana AU - Carbonell, Ruben G. T2 - SEPARATION AND PURIFICATION TECHNOLOGY AB - A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate. DA - 2023/7/15/ PY - 2023/7/15/ DO - 10.1016/j.seppur.2023.123920 VL - 317 SP - SN - 1873-3794 KW - Salt-tolerant multimodal ligand KW - Membrane adsorber KW - Membrane chromatography KW - Nonwoven membranes KW - Monoclonal antibodies (mAbs) KW - High-risk HCPs ER - TY - JOUR TI - Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography AU - Shastry, Shriarjun AU - WENNING, CHU AU - Barbieri, Eduardo AU - Greback-Clarke, Paul AU - Smith, William AU - Cummings, Christopher AU - Minzoni, Ariann AU - Pancorbo, Jenifer AU - Gilleskie, Gary AU - Ritola, Kimberly AU - Daniele, Michael AU - Menegatti, Stefano AB - Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. The AAV purification pipeline, in particular, hinges on protein ligands for the affinity-based capture step: while featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product’s activity and stability; additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable – yet equally effective – affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, while (ii) enabling product elution under near-physiological conditions (pH 6.0) and (iii) granting extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades – AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 – corroborating the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (~1014 vp per mL) and product yields (~60-80%) on par with commercial adsorbents, and purified AAV2 from HEK293 and Sf9 cell lysates affording high recovery (up to 78%) and reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%) of the purified vectors. DA - 2023/5/23/ PY - 2023/5/23/ DO - 10.22541/au.168482798.87003310/v1 UR - https://doi.org/10.22541/au.168482798.87003310/v1 ER - TY - JOUR TI - Development and Delivery of a Hands-On Short Course in Adeno-Associated Virus Manufacturing to Support Growing Workforce Needs in Gene Therapy AU - Overton, Laurie AU - Boi, Cristiana AU - Shastry, Shriarjun AU - Smith-Moore, Caroline AU - Balchunas, John AU - Sambandan, Deepa AU - Gilleskie, Gary T2 - HUMAN GENE THERAPY AB - The manufacturing of gene therapy products is a rapidly growing industry bolstered by the tremendous potential of these therapies to provide lifesaving treatment for rare and complex genetic diseases. The industry's steep rise has resulted in a high demand for skilled staff required to manufacture gene therapy products of the expected high quality. To address this skill shortage, more opportunities for education and training in all aspects of gene therapy manufacturing are needed. The Biomanufacturing Training and Education Center (BTEC) at the North Carolina State University (NC State) has developed and delivered (and continues to deliver) a 4-day, hands-on course titled Hands-on cGMP Biomanufacturing of Vectors for Gene Therapy. The course, which consists of 60% hands-on laboratory activities and 40% lectures, aims to provide a comprehensive understanding of the gene therapy production process, from vial thaw through the final formulation step, and analytical testing. This article discusses the design of the course, the backgrounds of the nearly 80 students who have participated in the seven offerings held since March 2019, and feedback from the course participants. DA - 2023/4/1/ PY - 2023/4/1/ DO - 10.1089/hum.2022.235 VL - 34 IS - 7-8 SP - 259-272 SN - 1557-7422 UR - http://dx.doi.org/10.1089/hum.2022.235 KW - viral vectors KW - AAV KW - gene therapy KW - biomanufacturing KW - training KW - short course ER - TY - JOUR TI - Investigation of a Listeria monocytogenes Chromosomal Immigration Control Region Reveals Diverse Restriction Modification Systems with Complete Sequence Type Conservation AU - Brown, Phillip AU - Lee, Sangmi AU - Elhanafi, Driss AU - Tham, Wilhelm AU - Danielsson-Tham, Marie-Louise AU - Lopez-Valladares, Gloria AU - Chen, Yi AU - Ivanova, Mirena AU - Leekitcharoenphon, Pimlapas AU - Kathariou, Sophia T2 - MICROORGANISMS AB - Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability. DA - 2023/3// PY - 2023/3// DO - 10.3390/microorganisms11030699 VL - 11 IS - 3 SP - SN - 2076-2607 KW - Listeria KW - restriction modification system KW - immigration control region KW - whole genome sequencing KW - chromosomal hotspot ER - TY - JOUR TI - Membrane-localized expression, production and assembly of Vibrio parahaemolyticus T3SS2 provides evidence for transertion AU - Kaval, Karan Gautam AU - Chimalapati, Suneeta AU - Siegel, Sara D. AU - Garcia, Nalleli AU - Jaishankar, Jananee AU - Dalia, Ankur B. AU - Orth, Kim T2 - NATURE COMMUNICATIONS AB - Abstract It has been proposed that bacterial membrane proteins may be synthesized and inserted into the membrane by a process known as transertion, which involves membrane association of their encoding genes, followed by coupled transcription, translation and membrane insertion. Here, we provide evidence supporting that the pathogen Vibrio parahaemolyticus uses transertion to assemble its type III secretion system (T3SS2), to inject virulence factors into host cells. We propose a two-step transertion process where the membrane-bound co-component receptor (VtrA/VtrC) is first activated by bile acids, leading to membrane association and expression of its target gene, vtrB , located in the T3SS2 pathogenicity island. VtrB, the transmembrane transcriptional activator of T3SS2, then induces the localized expression and membrane assembly of the T3SS2 structural components and its effectors. We hypothesize that the proposed transertion process may be used by other enteric bacteria for efficient assembly of membrane-bound molecular complexes in response to extracellular signals. DA - 2023/3/2/ PY - 2023/3/2/ DO - 10.1038/s41467-023-36762-z VL - 14 IS - 1 SP - SN - 2041-1723 ER - TY - JOUR TI - Immunological and Oxidative Biomarkers in Bovine Serum from Healthy, Clinical, and Sub-Clinical Mastitis Caused by Escherichia coli and Staphylococcus aureus Infection AU - Sadat, Asmaa AU - Farag, Alshimaa M. M. AU - Elhanafi, Driss AU - Awad, Amal AU - Elmahallawy, Ehab Kotb AU - Alsowayeh, Noorah AU - El-khadragy, Manal F. AU - Elshopakey, Gehad E. T2 - ANIMALS AB - The study aimed to investigate the mastitis' emerging causative agents and their antimicrobial sensitivity, in addition to the hematological, biochemical indicators, oxidative biomarkers, acute phase protein (APP), and inflammatory cytokine changes in dairy farms in Gamasa, Dakahlia Governorate, Egypt. One hundred Holstein Friesian dairy cattle with clinical and subclinical mastitis were investigated and were allocated into three groups based on a thorough clinical examination. Escherichia coli and Staphylococcus aureus were found responsible for the clinical and subclinical mastitis in dairy farms, respectively. Multiple drug resistance (MDR) was detected in 100%, and 94.74% of E. coli and S. aureus isolates, respectively. Significantly low RBCs count, Hb, and PCV values were detected in mastitic cows compared with both subclinical mastitic and control groups; moreover, WBCs, lymphocytes, and neutrophil counts were significantly diminished in mastitic cows compared to the controls. Significantly higher levels of AST, LDH, total protein, and globulin were noticed in both mastitic and subclinical mastitic cows. The haptoglobin, fibrinogen, amyloid A, ceruloplasmin, TNF-α, IL-1β, and IL-6 levels were statistically increased in mastitic cows compared to the controls. Higher MDA levels and reduction of TAC and catalase were identified in all the mastitic cases compared to the controls. Overall, the findings suggested potential public health hazards due to antimicrobial resistance emergence. Meanwhile, the APP and cytokines, along with antioxidant markers can be used as early indicators of mastitis. DA - 2023/3// PY - 2023/3// DO - 10.3390/ani13050892 VL - 13 IS - 5 SP - SN - 2076-2615 KW - mastitis KW - E KW - coli KW - S KW - aureus KW - oxidative KW - antioxidant molecules KW - APP KW - inflammatory cytokines ER - TY - JOUR TI - Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates AU - Chu, Wenning AU - Barbieri, Eduardo AU - Prodromou, Raphael AU - Greback-Clarke, Paul AU - Smith, William K AU - Moore, Brandyn D AU - Kilgore, Ryan E AU - Cummings, Christopher L AU - Pancorbo, Jennifer AU - Gilleskie, Gary L AU - Daniele, Michael A AU - Menegatti, Stefano AB - Abstract Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands – typically camelid antibodies – that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (K D ∼ 10 - 5 -10 -6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC 10% > 10 13 vp per mL of resin) and product yields (∼50-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50-80%), 80-to-400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses. DA - 2023/2/21/ PY - 2023/2/21/ DO - 10.1101/2023.02.19.529155 UR - https://doi.org/10.1101/2023.02.19.529155 ER - TY - JOUR TI - Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids AU - Kilgore, Ryan AU - Chu, Wenning AU - Bhandari, Dipendra AU - Fischler, David AU - Carbonell, Ruben G. AU - Crapanzano, Michael AU - Menegatti, Stefano T2 - JOURNAL OF CHROMATOGRAPHY A AB - Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10-6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93-96%) and purity (89-96%) comparable to those provided by Protein L resins. DA - 2023/1/4/ PY - 2023/1/4/ DO - 10.1016/j.chroma.2022.463701 VL - 1687 SP - SN - 1873-3778 KW - Peptide ligands KW - Affinity chromatography KW - Fragment antigen binding KW - Monoclonal antibodies KW - Cell culture harvests ER - TY - JOUR TI - Integrated in silico and experimental discovery of trimeric peptide ligands targeting Butyrylcholinesterase AU - Mukherjee, Rudra Palash AU - Yow, Geok-Yong AU - Sarakbi, Samuel AU - Menegatti, Stefano AU - V. Gurgel, Patrick AU - Carbonell, Ruben G. AU - Bobay, Benjamin G. T2 - COMPUTATIONAL BIOLOGY AND CHEMISTRY AB - Butyrylcholinesterase (BChE) is recognized as a high value biotherapeutic in the treatment of Alzheimer's disease and drug addiction. This study presents the rational design and screening of an in-silico library of trimeric peptides against BChE and the experimental characterization of peptide ligands for purification. The selected peptides consistently afforded high BChE recovery (> 90 %) and purity, yielding up to a 1000-fold purification factor. This study revealed a marked anti-correlated conformational movement governed by the ionic strength and pH of the aqueous environment, which ultimately controls BChE binding and release during chromatographic purification; and highlighted the role of residues within and allosteric to the catalytic triad of BChE in determining biorecognition, thus providing useful guidance for ligand design and affinity maturation. DA - 2023/2// PY - 2023/2// DO - 10.1016/j.compbiolchem.2022.107797 VL - 102 SP - SN - 1476-928X KW - Acetylcholinesterase KW - Butyrylcholinesterase KW - Purification KW - Molecular docking KW - Affinity ligands ER -