Immunogenic Potential of DNA Vaccine candidate, ZyCoV-D against SARS-CoV-2 in Animal Models

2021 ◽  
Author(s):  
Ayan Dey ◽  
T.M. Chozhavel Rajanathan ◽  
Harish Chandra ◽  
Hari P.R. Pericherla ◽  
Sanjeev Kumar ◽  
...  
Keyword(s):  
Dna Vaccine ◽  
Neutralizing Antibodies ◽  
Animal Studies ◽  
Large Scale ◽  
Rabbit Model ◽  
Plasmid Vector ◽  
Protein S ◽  
Scale Production ◽  
Gene Encoding ◽  
Dna Plasmid

AbstractSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500μg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also provided Th-1 response as evidenced by elevated IFN-γ levels.

scholarly journals mRNA based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus neutralizing antibodies and mediates protection in rodents

2020 ◽  
Author(s):  
Susanne Rauch ◽  
Nicole Roth ◽  
Kim Schwendt ◽  
Mariola Fotin-Mleczek ◽  
Stefan O. Mueller ◽  
...  
Keyword(s):  
Viral Replication ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Protein S ◽  
Scale Production ◽  
Favourable Safety ◽  
Favourable Safety Profile ◽  
Humoral Responses

AbstractThe devastating SARS-CoV-2 pandemic demands rapid vaccine development and large scale production to meet worldwide needs. mRNA vaccines have emerged as one of the most promising technologies to address this unprecedented challenge. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle (LNP) encapsulated non-modified mRNA vaccine that encodes the full length, pre-fusion stabilised SARS-CoV-2 Spike (S) protein. S translated from CVnCoV is cleaved, post-translationally modified, and presented on the cell surface, highlighting the ability of mRNA vaccines to mimic antigen presentation during viral infection. Immunisation with CVnCoV induced strong humoral responses with high titres of virus neutralizing antibodies in mice and hamsters and robust CD4+ and CD8+ T cell responses in mice. Most importantly, vaccination with CVnCoV fully protected hamster lungs from challenge with wild type SARS-CoV-2. To gain insights in the risk of vaccine-enhanced disease, hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication were analysed for signs of vaccine-enhanced disease. No evidence of increased viral replication or exacerbated inflammation and damage to viral target organs was detectable, giving strong evidence for a favourable safety profile of CVnCoV. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.

scholarly journals SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Nikolaos C. Kyriakidis ◽  
Andrés López-Cortés ◽  
Eduardo Vásconez González ◽  
Alejandra Barreto Grimaldos ◽  
Esteban Ortiz Prado
Keyword(s):  
Neutralizing Antibodies ◽  
Large Scale ◽  
Vaccine Development ◽  
Rna Virus ◽  
Protein S ◽  
Effective Vaccine ◽  
Phase 3 ◽  
Vaccine Candidates ◽  
Advantages And Disadvantages ◽  
The World

AbstractThe new SARS-CoV-2 virus is an RNA virus that belongs to the Coronaviridae family and causes COVID-19 disease. The newly sequenced virus appears to originate in China and rapidly spread throughout the world, becoming a pandemic that, until January 5th, 2021, has caused more than 1,866,000 deaths. Hence, laboratories worldwide are developing an effective vaccine against this disease, which will be essential to reduce morbidity and mortality. Currently, there more than 64 vaccine candidates, most of them aiming to induce neutralizing antibodies against the spike protein (S). These antibodies will prevent uptake through the human ACE-2 receptor, thereby limiting viral entrance. Different vaccine platforms are being used for vaccine development, each one presenting several advantages and disadvantages. Thus far, thirteen vaccine candidates are being tested in Phase 3 clinical trials; therefore, it is closer to receiving approval or authorization for large-scale immunizations.

scholarly journals Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

2008 ◽  
Vol 74 (10) ◽  
pp. 2967-2975 ◽  
Author(s):  
Ryan D. Woodyer ◽  
Nathan J. Wymer ◽  
F. Michael Racine ◽  
Shama N. Khan ◽  
Badal C. Saha
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Active Form ◽  
Apium Graveolens ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
One Step ◽  
Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

Preparation, Characterization, and Biodistribution of Glutathione PEGylated Nanoliposomal Doxorubicin for Brain Drug Delivery with A Post-insertion Approach

2021 ◽  
Author(s):  
Amin mehrabian ◽  
Roghayyeh Vakili-Ghartavol ◽  
Mohammad Mashreghi ◽  
Sara Shokooh Saremi ◽  
Ali Badiee ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Animal Studies ◽  
Large Scale ◽  
Fluorescent Microscopy ◽  
Pegylated Liposomal Doxorubicin ◽  
Brain Barrier ◽  
Pharmacokinetic Studies ◽  
Scale Production ◽  
Insertion Technique ◽  
Blood Brain

Abstract Brain cancer treatments have been largely unsuccessful due to the blood-brain barrier. Several publications support the presence of glutathione (GSH) receptors on the surface of the BBB and consequently the products such as the 2B3-101, which is almost 5% pre-inserted GSH PEGylated liposomal doxorubicin, is under process in clinical studies. Here we conducted the PEGylated nanoliposomal doxorubicin particles that are covalently attached to the glutathione using the post-insertion technique. The post-insertion methodology is noticeably simpler, faster, and more cost-effective compared to the pre-insertion method which makes it desirable for large-scale pharmaceutical manufacturing. The 25, 50, 100, 200, and 400 ligands of the DSPE PEG(2000) Maleimide-GSH complexes were incorporated into the available Caelyx. According to the animal studies such as biodistribution, fluorescent microscopy, and pharmacokinetic studies, the 200L and 400L treatment arms were the most promising formulations compared to the Caelyx. They proved that post-inserted nanocarriers with 40 times lower levels of GSH micelles compared to the 2B3-101 have significantly increased the penetrance through the blood-brain barrier. Other tissue analysis showed that the doxorubicin will likely accumulate in the liver, spleen, heart, and lung in comparison with the Caelyx due to the expressed GSH receptors on tissues as an endogenous antioxidant. In conclusion, as was expected, the post-insertion technique was found a successful approach with more pharmaceutical aspects for large-scale production. Moreover, it is highly recommended further investigations to determine the efficacy of 5% post-inserted GSH targeted nanoliposomes versus the 2B3-101 as a similar formulation with a different preparation method.

scholarly journals Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Keyword(s):  
Pichia Pastoris ◽  
Receptor Binding ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Binding Domain ◽  
Size Exclusion ◽  
Scale Production ◽  
Receptor Binding Domain ◽  
Serological Tests

AbstractThe yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

scholarly journals Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

2004 ◽  
Vol 70 (6) ◽  
pp. 3292-3297 ◽  
Author(s):  
Gerard M. Gibbs ◽  
Barrie E. Davidson ◽  
Alan J. Hillier
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression System ◽  
Reversed Phase ◽  
Scale Production ◽  
Gene Encoding ◽  
Strong Activity ◽  
E Coli ◽  
Large Scale Production ◽  
Bacterial Cell Membrane

ABSTRACT Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

scholarly journals Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Franz X. Heinz ◽  
Karin Stiasny
Keyword(s):  
Neutralizing Antibodies ◽  
Large Scale ◽  
Adenovirus Vector ◽  
Antigen Expression ◽  
Protein S ◽  
Antibody Responses ◽  
Structural Modifications ◽  
Market Authorization ◽  
A Subunit ◽  
Distinguishing Features

AbstractCOVID-19 vaccines were developed with an unprecedented pace since the beginning of the pandemic. Several of them have reached market authorization and mass production, leading to their global application on a large scale. This enormous progress was achieved with fundamentally different vaccine technologies used in parallel. mRNA, adenoviral vector as well as inactivated whole-virus vaccines are now in widespread use, and a subunit vaccine is in a final stage of authorization. They all rely on the native viral spike protein (S) of SARS-CoV-2 for inducing potently neutralizing antibodies, but the presentation of this key antigen to the immune system differs substantially between the different categories of vaccines. In this article, we review the relevance of structural modifications of S in different vaccines and the different modes of antigen expression after vaccination with genetic adenovirus-vector and mRNA vaccines. Distinguishing characteristics and unknown features are highlighted in the context of protective antibody responses and reactogenicity of vaccines.

scholarly journals Tolerance engineering in Deinococcus geothermalis by heterologous efflux pumps

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Erika Boulant ◽  
Emmanuelle Cambon ◽  
Julia Vergalli ◽  
Rémi Bernard ◽  
Fabienne Neulat-Ripoll ◽  
...  
Keyword(s):  
Large Scale ◽  
Efflux Pumps ◽  
Toxic Effects ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
Deinococcus Geothermalis ◽  
Genes Encoding ◽  
Susceptibility Tests

AbstractProducing industrially significant compounds with more environmentally friendly represents a challenging task. The large-scale production of an exogenous molecule in a host microfactory can quickly cause toxic effects, forcing the cell to inhibit production to survive. The key point to counter these toxic effects is to promote a gain of tolerance in the host, for instance, by inducing a constant flux of the neo-synthetized compound out of the producing cells. Efflux pumps are membrane proteins that constitute the most powerful mechanism to release molecules out of cells. We propose here a new biological model, Deinococcus geothermalis, organism known for its ability to survive hostile environment; with the aim of coupling the promising industrial potential of this species with that of heterologous efflux pumps to promote engineering tolerance. In this study, clones of D. geothermalis containing various genes encoding chromosomal heterologous efflux pumps were generated. Resistant recombinants were selected using antibiotic susceptibility tests to screen promising candidates. We then developed a method to determine the efflux efficiency of the best candidate, which contains the gene encoding the MdfA of Salmonella enterica serovar Choleraesuis. We observe 1.6 times more compound in the external medium of the hit recombinant than that of the WT at early incubation time. The data presented here will contribute to better understanding of the parameters required for efficient production in D. geothermalis.

scholarly journals Enhanced ability of plant-derived PGT121 glycovariants to eliminate HIV-1-infected cells

2021 ◽  
Author(s):  
Sai Priya Anand ◽  
Shilei Ding ◽  
William D. Tolbert ◽  
Jérémie Prévost ◽  
Jonathan Richard ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Scale Up ◽  
Scale Production ◽  
Viral Neutralization ◽  
Antiviral Activities ◽  
Infected Cells ◽  
Hiv 1

The activity of broadly neutralizing antibodies (bNAbs) targeting HIV-1 depends on pleiotropic functions including viral neutralization and the elimination of HIV-1-infected cells. Several in vivo studies have suggested that passive administration of bNAbs represents a valuable strategy for the prevention or treatment of HIV-1. Additionally, different strategies are currently being tested to scale-up the production of bNAbs to obtain the large quantities of antibodies required for clinical trials. Production of antibodies in plants permits low-cost and large-scale production of valuable therapeutics; furthermore, pertinent to this work, it also includes an advanced glycoengineering platform. In this study, we used Nicotiana benthamiana to produce different Fc-glycovariants of a potent bNAb, PGT121, with near-homogeneous profiles and evaluated their antiviral activities. Structural analyses identified a close similarity in overall structure and glycosylation patterns of Fc regions for these plant-derived Abs and mammalian cell-derived Abs. When tested for Fc-effector activities, afucosylated PGT121 showed significantly enhanced FcγRIIIa interaction and antibody dependent cellular cytotoxicity (ADCC) against primary HIV-1-infected cells, both in vitro and ex vivo . However, the overall galactosylation profiles of plant PGT121 did not affect ADCC activities against infected primary CD4+ T cells. Our results suggest that the abrogation of the Fc N-linked glycan fucosylation of PGT121 is a worthwhile strategy to boost its Fc-effector functionality. IMPORTANCE PGT121 is a highly potent bNAb and its antiviral activities for HIV-1 prevention and therapy are currently being evaluated in clinical trials. The importance of its Fc-effector functions in clearing HIV-1-infected cells is also under investigation. Our results highlight enhanced Fc-effector activities of afucosylated PGT121 mAbs that could be important in a therapeutic context to accelerate infected cell clearance and slow disease progression. Future studies to evaluate the potential of plant-produced afucosylated PGT121 in controlling HIV-1 replication in vivo are warranted.

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