ABSTRACT
The eradication of smallpox was officially declared by the WHO in 1980, leading to discontinuation of the vaccination campaign against the virus. Consequently, immunity against smallpox and related orthopoxviruses like Monkeypox virus gradually declines, highlighting the need for efficient countermeasures not only for the prevention, but also for the treatment of already exposed individuals. We have recently developed human-like monoclonal antibodies (mAbs) from vaccinia virus-immunized non-human primates. Two mAbs, MV33 and EV42, targeting the two infectious forms of the virus, were selected for in vivo evaluation, based on their in vitro neutralization potency. A single dose of either MV33 or EV42 administered three days post-infection (dpi) to BALB/c female mice provides full protection against lethal ectromelia virus challenge. Importantly, a combination of both mAbs confers full protection even when provided five dpi. Whole-body bioimaging and viral load analysis reveal that combination of the two mAbs allows for faster and more efficient clearance of the virus from target organs compared to either MV33 or EV42 separately. The combined mAbs treatment further confers post-exposure protection against the currently circulating Monkeypox virus in Cast/EiJ female mice, highlighting their therapeutic potential against other orthopoxviruses.
Subject(s)
Orthopoxvirus , Poxviridae Infections , Smallpox , Vaccinia , Humans , Female , Animals , Mice , Antibodies, Monoclonal , Poxviridae Infections/prevention & control , Vaccinia virus , Antibodies, ViralABSTRACT
The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than seven million deaths worldwide. To reduce viral spread, the Israel Institute for Biological Research (IIBR) developed and produced a new rVSV-SARS-CoV-2-S vaccine candidate (BriLife®) based on a platform of a genetically engineered vesicular stomatitis virus (VSV) vector that expresses the spike protein of SARS-CoV-2 instead of the VSV-G protein on the virus surface. Quantifying the virus titer to evaluate vaccine potency requires a reliable validated assay that meets all the stringent pharmacopeial requirements of a bioanalytical method. Here, for the first time, we present the development and extensive validation of a quantitative plaque assay using Vero E6 cells for the determination of the concentration of the rVSV-SARS-CoV-2-S viral vector. Three different vaccine preparations with varying titers (DP_low, DP_high, and QC sample) were tested according to a strict validation protocol. The newly developed plaque assay was found to be highly specific, accurate, precise, and robust. The mean deviations from the predetermined titers for the DP_low, DP_high, and QC preparations were 0.01, 0.02, and 0.09 log10, respectively. Moreover, the mean %CV values for intra-assay precision were 18.7%, 12.0%, and 6.0%, respectively. The virus titers did not deviate from the established values between cell passages 5 and 19, and no correlation was found between titer and passage. The validation results presented herein indicate that the newly developed plaque assay can be used to determine the concentration of the BriLife® vaccine, suggesting that the current protocol is a reliable methodology for validating plaque assays for other viral vaccines.
ABSTRACT
The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms.
ABSTRACT
Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.
ABSTRACT
Hyper-immune antisera from large mammals, in particular horses, are routinely used for life-saving anti-intoxication intervention. While highly efficient, the use of these immunotherapeutics is complicated by possible recipient reactogenicity and limited availability. Accordingly, there is an urgent need for alternative improved next-generation immunotherapies to respond to this issue of high public health priority. Here, we document the development of previously unavailable tools for equine antibody engineering. A novel primer set, EquPD v2020, based on equine V-gene data, was designed for efficient and accurate amplification of rearranged horse antibody V-segments. The primer set served for generation of immune phage display libraries, representing highly diverse V-gene repertoires of horses immunized against botulinum A or B neurotoxins. Highly specific scFv clones were selected and expressed as full-length antibodies, carrying equine V-genes and human Gamma1/Lambda constant genes, to be referred as "Centaur antibodies". Preliminary assessment in a murine model of botulism established their therapeutic potential. The experimental approach detailed in the current report, represents a valuable tool for isolation and engineering of therapeutic equine antibodies.
Subject(s)
Antibodies , Immunoglobulin Variable Region , Animals , Antibodies/genetics , Cell Surface Display Techniques , Horses , Humans , Immunoglobulin Variable Region/genetics , Mammals , Mice , Neurotoxins , Recombinant Proteins/geneticsABSTRACT
Recent advances in the field of cell therapy have proposed new solutions for tissue repair and regeneration using various cell delivery approaches. Here we studied ex vivo a novel topical delivery system of encapsulated cells in hybrid polyethylene glycol-fibrinogen (PEG-Fb) hydrogel microspheres to respiratory tract models. We investigated basic parameters of cell encapsulation, delivery and release in conditions of inflamed and damaged lungs of bacterial-infected mice. The establishment of each step in the study was essential for the proof of concept. We demonstrated co-encapsulation of alveolar macrophages and epithelial cells that were highly viable and equally distributed inside the microspheres. We found that encapsulated macrophages exposed to bacterial endotoxin lipopolysaccharide preserved high viability and secreted moderate levels of TNFα, whereas non-encapsulated cells exhibited a burst TNFα secretion and reduced viability. LPS-exposed encapsulated macrophages exhibited elongated morphology and out-migration capability from microspheres. Microsphere degradation and cell release in inflamed lung environment was studied ex vivo by the incubation of encapsulated macrophages with lung extracts derived from intranasally infected mice with Yersinia pestis, demonstrating the potential in cell targeting and release in inflamed lungs. Finally, we demonstrated microsphere delivery to a multi-component airways-on-chip platform that mimic human nasal, bronchial and alveolar airways in serially connected compartments. This study demonstrates the feasibility in using hydrogel microspheres as an effective method for topical cell delivery to the lungs in the context of pulmonary damage and the need for tissue repair.
ABSTRACT
The Ambr15 system is an automated, high-throughput bioreactor platform which comprises 24 individually controlled, single-use stirred-tank reactors. This system plays a critical role in process development by reducing reagent requirements and facilitating high-throughput screening of process parameters. However, until now, the system was used to simulate processes involving cells in suspension or growing on microcarriers and has never been tested for simulating cells growing on macrocarriers. Moreover, to our knowledge, a complete production process including cell growth and virus production has never been simulated. Here, we demonstrate, for the first time, the amenability of the automated Ambr15 cell culture reactor system to simulate the entire SARS-CoV-2 vaccine production process using macrocarriers. To simulate the production process, accessories were first developed to enable insertion of tens of Fibra-Cel macrocarries into the reactors. Vero cell adsorption to Fibra-Cels was then monitored and its adsorption curve was studied. After incorporating of all optimized factors, Vero cells were adsorbed to and grown on Fibra-Cels for several days. During the process, culture medium was exchanged, and the quantity and viability of the cells were followed, resulting in a typical growth curve. After successfully growing cells for 6 days, they were infected with the rVSV-ΔG-Spike vaccine virus. The present results indicate that the Ambr15 system is not only suitable for simulating a process using macrocarriers, but also to simulate an entire vaccine production process, from cell adsorption, cell growth, infection and vaccine virus production.
Subject(s)
COVID-19 , Virus Cultivation , Animals , Bioreactors , COVID-19/prevention & control , COVID-19 Vaccines , Cell Culture Techniques/methods , Chlorocebus aethiops , Humans , SARS-CoV-2 , Vero Cells , Virus Cultivation/methodsABSTRACT
Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes.
ABSTRACT
To face the coronavirus disease 2019 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, our institute has developed the rVSV-ΔG-spike vaccine, in which the glycoprotein of vesicular stomatitis virus (VSV) was replaced by the spike protein of SARS-CoV-2. Many process parameters can influence production yield. To maximize virus vaccine yield, each parameter should be tested independently and in combination with others. Here, we report the optimization of the production of the VSV-ΔG-spike vaccine in Vero cells using the Ambr15 system. This system facilitates high-throughput screening of process parameters, as it contains 24 individually controlled, single-use stirred-tank minireactors. During optimization, critical parameters were tested. Those parameters included: cell densities; the multiplicity of infection; virus production temperature; medium addition and medium exchange; and supplementation of glucose in the virus production step. Virus production temperature, medium addition, and medium exchange were all found to significantly influence the yield. The optimized parameters were tested in the BioBLU 5p bioreactors production process and those that were found to contribute to the vaccine yield were integrated into the final process. The findings of this study demonstrate that an Ambr15 system is an effective tool for bioprocess optimization of vaccine production using macrocarriers and that the combination of production temperature, rate of medium addition, and medium exchange significantly improved virus yield.
Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Chlorocebus aethiops , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Vero CellsABSTRACT
The spike glycoprotein mediates virus binding to the host cells and is a key target for vaccines development. One SARS-CoV-2 vaccine is based on vesicular stomatitis virus (VSV), in which the native surface glycoprotein has been replaced by the SARS-CoV-2 spike protein (VSV-ΔG-spike). The titer of the virus is quantified by the plaque forming unit (PFU) assay, but there is no method for spike protein quantitation as an antigen in a VSV-based vaccine. Here, we describe a mass spectrometric (MS) spike protein quantification method, applied to VSV-ΔG-spike based vaccine. Proof of concept of this method, combining two different sample preparations, is shown for complex matrix samples, produced during the vaccine manufacturing processes. Total spike levels were correlated with results from activity assays, and ranged between 0.3-0.5 µg of spike protein per 107 PFU virus-based vaccine. This method is simple, linear over a wide range, allows quantification of antigen within a sample and can be easily implemented for any vaccine or therapeutic sample.
Subject(s)
COVID-19 , Viral Vaccines , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Mass Spectrometry , SARS-CoV-2 , Spike Glycoprotein, CoronavirusABSTRACT
Polyvinyl alcohol (PVA)-based hydrogels are promising biomaterials for tissue engineering printing applications. However, one of their main disadvantages is their inability to support cell attachment, which is a critical feature for the preparation of biological scaffolds. The goal of this study was to develop a printable, cell-supportive PVA-based bioink with tunable mechanical properties, without using animal-derived polymers which potentially harbor human pathogens. An ultraviolet light (UV) curable PVA-methacrylate (PVA-MA) polymer mixed with Cys-Arg-Gly-Asp (CRGD) peptide was developed. This peptide holds the integrin receptor binding sequence - RGD, that can enhance cell attachment. The additional cysteine was designed to enable its thiol binding under UV to methacrylate groups of the UV curable PVA-MA. Vero cell, as an adherent cell model was used to assess the hydrogel's cell adhesion. It was found that the PVA-MA-CRGD formula enables the preparation of hydrogels with excellent cell attachment and had even shown superior cell attachment properties relative to added gelatin. Adding hyaluronic acid (HA) as a rheologic modulator enabled the printing of this new formula. Our overall data demonstrates the applicability of this mixture as a bioink for soft tissue engineering such as skin, adipose, liver or kidney tissue.
Subject(s)
Hydrogels , Polyvinyl Alcohol , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Humans , Hydrogels/chemistry , Hydrogels/pharmacology , Methacrylates/chemistry , Methacrylates/pharmacology , Peptides, Cyclic , Polyvinyl Alcohol/chemistry , Polyvinyl Alcohol/pharmacology , Tissue EngineeringABSTRACT
The pharmacopeia mouse neutralization assay (PMNA) is the standard method for determining the potency of pharmaceutical botulinum antitoxins. However, a PMNA requires a large number of mice, and, thus, an alternative in vitro method to replace it is needed. Herein, we developed an in vitro SiMa cell line-based neutralization assay (SBNA), compatible with a PMNA design, for therapeutic antitoxins against type E botulinum neurotoxin (BoNT/E). The SBNA measures the residual cellular activity of BoNT/E following antitoxin neutralization in the SiMa lysate using a specific quantitative sandwich ELISA for its cleaved cellular target protein SNAP-25. The potencies of different pharmaceutical antitoxin preparations were determined by applying two different quantification approaches: (1) a cutoff value, in accordance with the pharmacopeia concept, and (2) nonlinear regression of a standard curve generated by serial dilutions of a standard antitoxin. Both approaches achieved accurate potencies compared to the PMNA (average %RE of ~16%). Furthermore, the SBNA was able to determine in vitro, for the first time, the accurate neutralizing activity (%RE ≤ 20) of next-generation equine and rabbit therapeutic antitoxins. Collectively, a high correlation between SBNA and PMNA results was obtained for all antitoxin preparations (r = 0.99, P < 0.0001 for the standard curve approach, and r = 0.97, p < 0.0001 for the cutoff approach). In conclusion, the SBNA can potentially replace the PMNA and markedly reduce the need for laboratory animals for the approval of botulinum antitoxin preparations.
Subject(s)
Antitoxins , Botulinum Toxins, Type A , Botulism , Pharmaceutical Preparations , Animal Testing Alternatives , Animals , Botulinum Antitoxin , Horses , Mice , RabbitsABSTRACT
The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.
ABSTRACT
rVSV-Spike (rVSV-S) is a recombinant viral vaccine candidate under development to control the COVID-19 pandemic and is currently in phase II clinical trials. rVSV-S induces neutralizing antibodies and protects against SARS-CoV-2 infection in animal models. Bringing rVSV-S to clinical trials required the development of a scalable downstream process for the production of rVSV-S that can meet regulatory guidelines. The objective of this study was the development of the first downstream unit operations for cell-culture-derived rVSV-S, namely, the removal of nucleic acid contamination, the clarification and concentration of viral harvested supernatant, and buffer exchange. Retaining the infectivity of the rVSV-S during the downstream process was challenged by the shear sensitivity of the enveloped rVSV-S and its membrane protruding spike protein. Through a series of screening experiments, we evaluated and established the required endonuclease treatment conditions, filter train composition, and hollow fiber-tangential flow filtration parameters to remove large particles, reduce the load of impurities, and concentrate and exchange the buffer while retaining rVSV-S infectivity. The combined effect of the first unit operations on viral recovery and the removal of critical impurities was examined during scale-up experiments. Overall, approximately 40% of viral recovery was obtained and the regulatory requirements of less than 10 ng host cell DNA per dose were met. However, while 86-97% of the host cell proteins were removed, the regulatory acceptable HCP levels were not achieved, requiring subsequent purification and polishing steps. The results we obtained during the scale-up experiments were similar to those obtained during the screening experiments, indicating the scalability of the process. The findings of this study set the foundation for the development of a complete downstream manufacturing process, requiring subsequent purification and polishing unit operations for clinical preparations of rVSV-S.
Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Antibodies, Neutralizing , Humans , Pandemics , SARS-CoV-2 , Spike Glycoprotein, CoronavirusABSTRACT
A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Antibody Affinity , COVID-19/therapy , COVID-19/virology , Epitopes/genetics , Epitopes/immunology , Humans , Immunization, Passive , Mice , Mice, Transgenic , Models, Molecular , Neutralization Tests , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Treatment Outcome , COVID-19 SerotherapyABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Drug Discovery , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Animals , Antiviral Agents/therapeutic use , Aurintricarboxylic Acid/pharmacology , Aurintricarboxylic Acid/therapeutic use , COVID-19/virology , Chlorocebus aethiops , Ellagic Acid/pharmacology , Ellagic Acid/therapeutic use , Heparin/pharmacology , Heparin/therapeutic use , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Protein Domains/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effectsABSTRACT
The most effective way to prevent and control infectious disease outbreak is through vaccines. The increasing use of vaccines has elevated the need to establish new manufacturing strategies. One of the major approaches is cell-based production, which creates a need for high cell density to enable higher cell production levels. This has led to development of the technology of cell carriers, including micro and macro cell carriers. To follow the production process, quantifying the number of cells on these carriers is required, as well as the tracking of their viability and proliferation. However, owing to various carriers' unique structures, tracking the cell's is challenging using current traditional assays that were originally developed for monolayers of adherent cells. The current "gold standard" method is counting cell nuclei, separating cells from the carrier, staining with crystal violet, and visually counting under a microscope. This method is tedious and counts both live and dead cells. A few other techniques were developed but were specific to the carrier type and involved specialized equipment. In this study, we describe a broadly ranging method for counting cells on carriers that was developed and employed as part of the development of severe acute respiratory syndrome coronavirus 2 vaccine. The method is based on the Alamar blue dye, a well-known, common marker for cell activity, and was found to be successful in tracking cell adsorption, cell growth, and viability on carriers. No separation of the cells from the carriers is needed, nor is any specialized equipment; the method is simple and rapid and provides comprehensive details necessary for process control of viral vaccine production in cells. This method can be easily implemented in any of a number of cell-based processes and other unique platforms for measuring the growth of encapsulated cells.
Subject(s)
COVID-19 Vaccines , COVID-19/metabolism , SARS-CoV-2/metabolism , Animals , COVID-19/pathology , Cell Count , Chlorocebus aethiops , Humans , Vero CellsABSTRACT
Neutralizing antibodies represent a valuable therapeutic approach to countermeasure the current COVID-19 pandemic. Emergence of SARS-CoV-2 variants emphasizes the notion that antibody treatments need to rely on highly neutralizing monoclonal antibodies (mAbs), targeting several distinct epitopes for circumventing therapy escape mutants. Previously, we reported efficient human therapeutic mAbs recognizing epitopes on the spike receptor-binding domain (RBD) of SARS-CoV-2. Here we report the isolation, characterization, and recombinant production of 12 neutralizing human mAbs, targeting three distinct epitopes on the spike N-terminal domain of the virus. Neutralization mechanism of these antibodies involves receptors other than the canonical hACE2 on target cells, relying both on amino acid and N-glycan epitope recognition, suggesting alternative viral cellular portals. Two selected mAbs demonstrated full protection of K18-hACE2 transgenic mice when administered at low doses and late post-exposure, demonstrating the high potential of the mAbs for therapy of SARS-CoV-2 infection.
ABSTRACT
Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches.
Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , COVID-19/virology , Epitope Mapping , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Female , Humans , Neutralization Tests , Rabbits , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
Public health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many "antigen" detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2's nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices' tendency to exhibit false positive results. In this work, we developed a novel alternative spike-based antigen assay, comprising four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike's S1 subunit. The assay's performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of novel antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct < 25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct < 25) while less specific (87% specificity). Despite being outperformed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results. Schematic representation of sample collection and analysis. The figure was created using BioRender.com.