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...

The threat of a smallpox-based bioterrorist event or a human monkeypox outbreak has heightened the importance of new, safe vaccine approaches for these pathogens to complement older poxviral vaccine platforms. As poxviruses are large, complex viruses, they present technological challenges for simple recombinant vaccine development where a multicomponent mixtures of vaccine antigens are likely important in protection. We report that a synthetic, multivalent, highly concentrated, DNA vaccine delivered by a minimally invasive, novel skin electroporation microarray can drive polyvalent immunity in macaques, and offers protection from a highly pathogenic monkeypox challenge. Such a diverse, high-titer antibody response produced against 8 different DNA-encoded antigens delivered simultaneously in microvolumes has not been previously described. These studies represent a significant improvement in the efficiency of the DNA vaccine platform, resulting in immune responses that mimic live viral infections, and would likely have relevance for vaccine design against complex human and animal pathogens.

The neutral protease gene of Bacillus subtilis has been cloned, and its nucleotide sequence has been determined. The cloned gene was used to create an in vitro-derived deletion mutation, which was used to replace the wild-type copy of the gene. This deletion, in combination with a deletion of the alkaline protease gene, completely abolished protease production. The loss of the proteases had no detectable effect on growth, morphology, or sporulation.

We have determined the sequences ofthe recombinant DNA inserts of three bacterial plasmid cDNA clones containing most of the rat a-fetoprotein mRNA. The resultant nucleotide sequence of a-fetoprotein was exhaustively compared to the nucleotide sequence of the mRNA encoding rat serum albumin. These two mRNAs have extensive homology (50%) throughout and the same intron locations. The amino acid sequence of rat a-fetoprotein has been deduced from the nucleotide sequence, and its comparison to rat serum albumin's amino acid sequence reveals a 34% homology. The regularly spaced positions of the cysteines found in serum albumin are conserved in rat a-fetoprotein, indicating that these two proteins may have a similar secondary folding structure. These homologies indicate that a-fetoprotein and serum albumin were derived by duplication of a common ancestral gene and constitute a gene family. a-Fetoprotein (AFP) and serum albumin are major plasma proteins both synthesized in mammalian liver parenchymal cells. AFP is also produced in the embryonic yolk sac (1) and is a single chain glycoprotein with a molecular weight of70,000 containing 4% carbohydrate (2-6). AFP synthesis is associated with developmental and neoplastic processes (7-9), and it is the dominant plasma protein of the mammalian fetus. After birth the concentration ofAFP decreases to a trace level in the serum of healthy adult mammals (10, 11). On the other hand, production of serum albumin, a single chain polypeptide with a molecular weight of66,000, increases severalfold after birth to become the predominant protein in the adult serum (7,12,13). This inverse relationship in expression of AFP and serum albumin and the physical similarities between these two proteins suggest that AFP may be the fetal analog of serum albumin.The developmental alterations in the expression of AFP and serum albumin are of interest as an example ofeukaryotic gene regulation (14). Analysis of the sequence and the structural organization ofthese two genes should elucidate the evolutionary relationship between AFP and serum albumin, and may also aid in understanding their regulation. The mRNA nucleotide sequence and the structural organization of the rat serum albumin gene have been established by sequence analysis (15)(16)(17). The amino acid sequence data available for AFP have been compared to those for serum albumin and a degree of internal homology has been shown to exist between these two proteins (18-20). The structural organizations ofthe mouse AFP and serum albumin genes have been estimated from electron micrographs of R-loops, and there is some similarity in this regard between the rat albumin gene and the mouse AFP and albumin genes (21,22). The mouse AFP amino acid sequence has been deduced from the nucleotide sequence of its mRNA. A comparison of this sequence to that of human and bovine albumin revealed a 32% homology and regularly spaced cysteines (23). These homologies suggest that AFP and serum albumin are related, possibly derived by the duplication of a c...