The coronavirus family member, SARS-CoV-2 has been identified as the causal agent for the pandemic viral pneumonia disease, COVID-19. At this time, no vaccine is available to control further dissemination of the disease. We have previously engineered a synthetic DNA vaccine targeting the MERS coronavirus Spike (S) protein, the major surface antigen of coronaviruses, which is currently in clinical study. Here we build on this prior experience to generate a synthetic DNA-based vaccine candidate targeting SARS-CoV-2 S protein. The engineered construct, INO-4800, results in robust expression of the S protein in vitro. Following immunization of mice and guinea pigs with INO-4800 we measure antigen-specific T cell responses, functional antibodies which neutralize the SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and biodistribution of SARS-CoV-2 targeting antibodies to the lungs. This preliminary dataset identifies INO-4800 as a potential COVID-19 vaccine candidate, supporting further translational study.
Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.
Mutations in the sacU region have a pleiotropic phenotype. Certain mutations designated sacU(Hy), for example, express degradative enzymes at high levels, are able to sporulate in the presence of glucose, have severely reduced transformation efficiencies, and are nonmotile. We isolated and sequenced the sacU gene region of Bacillus subtilis. Two open reading frames were found in the sacU region, and sacU(Hy) mutations were localized to both of these open reading frames. The two open reading frames have similarities to two widespread families of proteins that mediate responses to environmental stimuli.In Bacillus subtilis, a variety of mutants have been isolated due to their ability to produce higher levels of particular degradative exoenzymes, such as amylase, proteases, or levansucrase (4,22,37). Upon further characterization, a number of these mutant strains were shown to produce higher levels of not only the original exoenzyme of interest but also most of the other exoenzymes examined (4, 21). The majority of the mutations were subsequently mapped to one genetic locus and are most commonly referred to as sacU(Hy) (40). These sacU(Hy) mutations have a pleiotropic phenotype. They overproduce a large number of enzymes that degrade polymeric substrates; these enzymes include alkaline and neutral protease, a-amylase, P-glucanase, levansucrase, and intracellular serine protease (1,23,33). Strains carrying these mutations can also sporulate efficiently in the presence of glucose, which wild-type strains of B. subtilis cannot do (23). These strains also lack flagella and are very poorly transformable (23). Other mutations, designated sacU, have also been mapped to the same location as the sacU(Hy) alleles or to a tightly linked location (22). These mutations almost completely abolish expression of the levansucrase and intracellular serine protease genes but have only a minimal effect on the expression of the other exoenzymes (22,33).The mechanism by which the sacU(Hy) mutations increase the level of expression of their target genes has been recently investigated. Studies of the levansucrase gene showed that strains carrying a sacU(Hy) mutation had an increased level of mRNA, and analysis of the mRNA by S1 analysis showed that the mRNA had the same transcriptional start site in sacU+ and sacU(Hy) strains (3, 39). A study of the alkaline protease gene showed similar results (12), suggesting that the stimulation of expression of these two genes is by increased transcription of their promoters. Deletion analysis of the alkaline protease and levansucrase promoters suggested that the target site for this stimulation in sacU(Hy) mutant strains is approximately 110 nucleotides upstream of the transcriptional start site (12, 13).Two other classes of regulatory mutants have been isolated which appear to have a phenotype similar to that of strains carrying the sacU(Hy) mutations. The sacQ and prtR * Corresponding author. genes encode 46-and 60-amino-acid proteins, respectively; overproduction of the sacQ or prtR polypeptides ap...
The gene coding for amylase (EC.3.2.1.1) has been isolated and sequenced from Bacillus subtilis by cloning in lambda Charon4A and pBR322. The entire coding sequence and large preceding and following regions, comprising the presumed transcriptional and translational regulatory regions, were sequenced. The coding sequence shows a large open reading frame with a translated molecular weight of 72,800 and a presumed signal sequence of approximately thirty-two amino acids. When the intact gene is present in Escherichia coli, it confers the ability to degrade starch, indicating that the gene is expressed in a functional state.
The sacQ gene of Bacillus subtilis, a pleiotropic gene affecting the expression of a number of secreted gene products, has been identified as a small 46-amino-acid polypeptide. The increased expression of this polypeptide in strains carrying the sacQ36 allele, or in strains carrying the sacQ gene on a high copy plasmid, appears to be responsible for the phenotype of higher levels of proteases seen in these strains. A deletion of the sacQ gene had no apparent phenotype, indicating that it is not an essential gene.A number of mutations have been isolated in the sucrose metabolism of Bacillus subtilis that cause the hyperproduction of the secreted enzyme levansucrase (20). Two loci, sacU and sacQ, were at first regarded as regulatory genes which played a specific role in the control of the levansucrase gene, sacB (20,21). However the strains carrying sacU and sacQ mutations are affected in the production of other extracellular enzymes, including amylase and proteases (19). Indeed, a number of mutations such as pap and amyB, which were defined on the basis of the overproduction of secreted amylase or protease, were shown to be identical to the sacU mutation (37).These mutations affect the production of genes which are regulated in very different fashions. For example, levansucrase is induced by growth in the presence of sucrose and is only produced during vegetative growth (11); however, amylases and the proteases are produced during postexponential growth (31). The only common feature of the genes whose production is affected by these pleiotropic mutations is that they are all secreted gene products, and an attractive theory is that they affect some component of the secretion system. Although a great deal of attention has focused on the ability of Bacillus species to secrete large quantities of industrial enzymes, very few studies have focused on the secretion process itself (8,9,31). Recently the sacU gene of B. subtilis has been isolated and shown to be a 46-kilodalton membrane protein (3). This report describes the isolation of the sacQ gene of B. subtilis, identifies its gene product as a 46-amino-acid polypeptide, and shows by deletion of the gene that it is nonessential for growth or secretion. MATERIALS AND METHODSBacterial strains, plasmids, and media. Escherichia coli MM294 (F-supE44 endAl thi-J hsdR4) was used as a host for plasmid constructions (4). Strain GM48 (dam-), received from H Boyer, was used to prepare plasmids that were subsequently digested with BclI. The B. subtilis strains used in this study are listed in Table 1. Amylase production was tested by growing colonies overnight on a nutrient broth plate containing 1% starch and staining the plate with iodine (34).Colonies were tested for P-galactosidase expression on minimal medium plates (36) containing 0.5% glucose, 0.1% casein hydrolysate and 50 p,g of 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside per ml. Plasmids pBR322 (7), pJF751 * Corresponding author. t Present address: Genencor, Inc., South San Francisco, CA 94080.(13), pC194 (17), pJ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.