scholarly journals Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Michael Logan ◽  
John Law ◽  
Jason Alexander Ji-Xhin Wong ◽  
Darren Hockman ◽  
Amir Landi ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Large Scale ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Affinity Purification ◽  
Scale Up ◽  
Vaccine Antigen ◽  
Scale Production ◽  
Genotype 1A ◽  
Neutralizing Epitopes

ABSTRACT A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

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 Development of Inactivated FAKHRAVAC® Vaccine against SARS-CoV-2 Virus: Preclinical Study in Animal Models

Vaccines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1271
Author(s):  
Soheil Ghasemi ◽  
Kosar Naderi Saffar ◽  
Firooz Ebrahimi ◽  
Pezhman Khatami ◽  
Arina Monazah ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Rhesus Macaques ◽  
Seroconversion Rate ◽  
Pilot Scale ◽  
Preclinical Study ◽  
Scale Production ◽  
Global Public Health ◽  
Health Crisis

The recent viral infection disease pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global public health crisis. Iran, as one of the countries that reported over five million infected cases by September 2021, has been concerned with the urgent development of effective vaccines against SARS-CoV-2. In this paper, we report the results of a study on potency and safety of an inactivated SARS-CoV-2 vaccine candidate (FAKHRAVAC) in a preclinical study so as to confirm its potential for further clinical evaluation. Here, we developed a pilot-scale production of FAKHRAVAC, a purified inactivated SARS-CoV-2 virus vaccine candidate that induces neutralizing antibodies in Balb/c mice, guinea pigs, rabbits, and non-human primates (Rhesus macaques—RM). After obtaining ethical code of IR.IUMS.REC.1399.566, immunizations of animals were conducted by using either of three different vaccine dilutions; High (H): 10 μg/dose, Medium (M): 5 μg/dose, and Low (L): 1 μg/dose, respectively. In the process of screening for viral seeds, viral strains that resulted in the most severe clinical manifestation in patients have been isolated for vaccine development. The viral seed produced the optimal immunity against SARS-CoV-2 virus, which suggests a possible broader neutralizing ability against SARS-CoV-2 strains. The seroconversion rate at the H-, M-, and L-dose groups of all tested animals reached 100% by 28 days after immunization. These data support the eligibility of FAKHRAVAC vaccine candidate for further evaluation in a clinical trial.

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.

scholarly journals Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii

2021 ◽  
Author(s):  
Neil C Dalvie ◽  
Andrew M Biedermann ◽  
Sergio A Rodriguez-Aponte ◽  
Christopher A Naranjo ◽  
Harish D Rao ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Large Scale ◽  
Scale Up ◽  
Binding Domain ◽  
Vaccine Antigen ◽  
Scale Production ◽  
Receptor Binding Domain ◽  
Methanol Induction ◽  
Vaccine Candidates ◽  
Komagataella Phaffii

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Solid Lipid Nanoparticles: A Promising Drug Delivery Technology

2009 ◽  
Vol 2 (2) ◽  
pp. 509-516
Author(s):  
S. Pragati ◽  
S. Kuldeep ◽  
S. Ashok ◽  
M. Satheesh
Keyword(s):  
Drug Delivery ◽  
Solid Lipid Nanoparticles ◽  
Large Scale ◽  
Colloidal Particles ◽  
Scale Up ◽  
Lipid Nanoparticles ◽  
Scale Production ◽  
Research Activity ◽  
New Approach ◽  
Solid Lipid

One of the situations in the treatment of disease is the delivery of efficacious medication of appropriate concentration to the site of action in a controlled and continual manner. Nanoparticle represents an important particulate carrier system, developed accordingly. Nanoparticles are solid colloidal particles ranging in size from 1 to 1000 nm and composed of macromolecular material. Nanoparticles could be polymeric or lipidic (SLNs). Industry estimates suggest that approximately 40% of lipophilic drug candidates fail due to solubility and formulation stability issues, prompting significant research activity in advanced lipophile delivery technologies. Solid lipid nanoparticle technology represents a promising new approach to lipophile drug delivery. Solid lipid nanoparticles (SLNs) are important advancement in this area. The bioacceptable and biodegradable nature of SLNs makes them less toxic as compared to polymeric nanoparticles. Supplemented with small size which prolongs the circulation time in blood, feasible scale up for large scale production and absence of burst effect makes them interesting candidates for study. In this present review this new approach is discussed in terms of their preparation, advantages, characterization and special features.

Recombinant Protein Production in Microalgae: Emerging Trends

2020 ◽  
Vol 27 (2) ◽  
pp. 105-110 ◽  
Author(s):  
Niaz Ahmad ◽  
Muhammad Aamer Mehmood ◽  
Sana Malik
Keyword(s):  
Chloroplast Genome ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Higher Plants ◽  
Scale Up ◽  
Chloroplast Transformation ◽  
Point Of View ◽  
Nuclear Transformation ◽  
Scale Production ◽  
Algal Chloroplast

: In recent years, microalgae have emerged as an alternative platform for large-scale production of recombinant proteins for different commercial applications. As a production platform, it has several advantages, including rapid growth, easily scale up and ability to grow with or without the external carbon source. Genetic transformation of several species has been established. Of these, Chlamydomonas reinhardtii has become significantly attractive for its potential to express foreign proteins inexpensively. All its three genomes – nuclear, mitochondrial and chloroplastic – have been sequenced. As a result, a wealth of information about its genetic machinery, protein expression mechanism (transcription, translation and post-translational modifications) is available. Over the years, various molecular tools have been developed for the manipulation of all these genomes. Various studies show that the transformation of the chloroplast genome has several advantages over nuclear transformation from the biopharming point of view. According to a recent survey, over 100 recombinant proteins have been expressed in algal chloroplasts. However, the expression levels achieved in the algal chloroplast genome are generally lower compared to the chloroplasts of higher plants. Work is therefore needed to make the algal chloroplast transformation commercially competitive. In this review, we discuss some examples from the algal research, which could play their role in making algal chloroplast commercially successful.

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 Scale-up of the production of highly reactive biogenic magnetite nanoparticles using Geobacter sulfurreducens

2015 ◽  
Vol 12 (107) ◽  
pp. 20150240 ◽  
Author(s):  
J. M. Byrne ◽  
H. Muhamadali ◽  
V. S. Coker ◽  
J. Cooper ◽  
J. R. Lloyd
Keyword(s):  
Magnetic Properties ◽  
Large Scale ◽  
Scale Up ◽  
Surface Reactivity ◽  
Geobacter Sulfurreducens ◽  
Scale Production ◽  
Buffer Solution ◽  
Pilot Plant Scale ◽  
Functional Biomaterials ◽  
Microbial Systems

Although there are numerous examples of large-scale commercial microbial synthesis routes for organic bioproducts, few studies have addressed the obvious potential for microbial systems to produce inorganic functional biomaterials at scale. Here we address this by focusing on the production of nanoscale biomagnetite particles by the Fe(III)-reducing bacterium Geobacter sulfurreducens , which was scaled up successfully from laboratory- to pilot plant-scale production, while maintaining the surface reactivity and magnetic properties which make this material well suited to commercial exploitation. At the largest scale tested, the bacterium was grown in a 50 l bioreactor, harvested and then inoculated into a buffer solution containing Fe(III)-oxyhydroxide and an electron donor and mediator, which promoted the formation of magnetite in under 24 h. This procedure was capable of producing up to 120 g of biomagnetite. The particle size distribution was maintained between 10 and 15 nm during scale-up of this second step from 10 ml to 10 l, with conserved magnetic properties and surface reactivity; the latter demonstrated by the reduction of Cr(VI). The process presented provides an environmentally benign route to magnetite production and serves as an alternative to harsher synthetic techniques, with the clear potential to be used to produce kilogram to tonne quantities.

scholarly journals Single-dose replicating RNA vaccine induces neutralizing antibodies against SARS-CoV-2 in nonhuman primates

2020 ◽  
Author(s):  
Jesse H. Erasmus ◽  
Amit P. Khandhar ◽  
Alexandra C. Walls ◽  
Emily A. Hemann ◽  
Megan A. O’Connor ◽  
...  
Keyword(s):  
Single Dose ◽  
Neutralizing Antibodies ◽  
Global Economy ◽  
Nonhuman Primates ◽  
Vaccine Candidate ◽  
Scale Up ◽  
Igg Antibody ◽  
Antibody Isotypes ◽  
Rna Replicon

AbstractThe ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.

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