Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine.

A DNA sequence coding for the immunogenic capsid protein VP3 of foot-and-mouth disease virus A12, prepared from the virion RNA, was ligated to a plasmid designed to express a chimeric protein from the Escherichia coli tryptophan promoter-operator system. When Escherichia coli transformed with this plasmid was grown in tryptophan-depleted media, approximately 17 percent of the total cellular protein was found to be an insoluble and stable chimeric protein. The purified chimeric protein competed equally on a molar basis with VP3 for specific antibodies to foot-and-mouth disease virus. When inoculated into six cattle and two swine, this protein elicited high levels of neutralizing antibody and protection against challenge with foot-and-mouth disease virus.

Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin.

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.

2-Deaminoactinomycin D, synthesis and interaction with deoxyribonucleic acid.

2-Deaminoactinomycin D has been synthesized and characterized. It binds to DNA by intercalation according to NMR, CD, thermal denaturation, and unwinding studies on the drug-DNA complex. Loss of the 2-amino group does not seriously affect binding parameters relative to actinomycin D; affinity for calf thymus DNA may even be increased, according to deltaTm measurements. The unwinding of circular DNA caused by this compound is at least as large as that effected by actinomycin D and ethidium bromide. Nevertheless, 2-deaminoactinomycin D is less effective than actinomycin D in inhibiting nucleic acid syntheses in L1210 cell culture and in in vivo antitumor activity against P388 leukemia.

Using genetically engineered bacteria for vaccine production.

We concluded from this and our earlier work that biosynthetically produced FMDV VP1-specific fusion proteins are effective vaccines. Whether this method of vaccine production can be extended to many other immunogenic proteins from other organisms is not known. Some problems that could be expected to occur with bacterially produced antigens are that the immunogenic site may not be properly exposed or the peptide sequence(s) within that site may not be able to form into the correct configuration. This could be caused by hydrophobic or hydrophilic interactions in the fusion protein that do not occur in the protein at the virus surface. Also, the immunogenic site may require disulfide bonding to bring two distant parts of a protein or two different peptide chains into close proximity to form an antigenic site, as demonstrated by the studies of Atassi et al. for lysozyme-using synthetic peptides. In summary, the use of genetically programmed bacteria is a promising avenue to vaccine manufacture. For FMD, biosynthetic protein vaccines have significant advantages over current whole-virus technology.

Dose-response evaluation of a genetically engineered foot-and-mouth disease virus polypeptide immunogen in cattle.

Four groups of 9 cattle each were vaccinated with 10, 50, 250, or 1,250 micrograms of foot-and-mouth disease (FMD) virus A12 VP1 fusion protein that was produced in Escherichia coli and emulsified in an oil adjuvant. The groups given the 10 and 50 micrograms of antigen were revaccinated at 15 weeks and were challenge exposed at 30 weeks; 5 of 9 and 7 of 9 cattle, respectively, were protected from FMD virus infection. The remaining 2 groups, vaccinated with 250 or 1,250 micrograms of antigen, were revaccinated at 32 weeks and were challenge exposed at 45 weeks; 8 of 9 and 9 of 9 cattle, respectively, were protected. The results indicated that the biosynthetic polypeptide FMD vaccine was effective using vaccination intervals frequently followed with conventional whole-virus vaccines.

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.

A method for classification of 5' termini of retroviruses.

A new method for the classification of retroviruses is presented. The scheme is based on the length and sequence of a DNA transcript of the 5' end of the genome. The method can be used to detect similarities between distantly related viruses as well as to discriminate between very closely related viruses. The method is applied to viruses isolated from mice, baboons, gibbons, a woolly monkey and chickens.

Lambda repressor turns off transcription of its own gene.

We report transcription in vitro of the lambda repressor gene, cI, using specific restriction endonuclease fragments as templates. This transcription is repressed by lambda repressor. Moreover, we report the sequence change caused by a cI promoter mutation. This change is located between two repressor binding sites in the rightward operator (OR). Transcription studies using mutant templates indicate that repressor bound to two sites in OR regulates transcription of gene tof, and repressor bound to the remaining site(s) controls transcription of cI.

Nucleotide sequence of the rightward operator of phage lambda.

The sequence of 72 base pairs of the rightward operator (O-R) of bacteriophage lambda is presented as determined with simple and rapid methods for direct DNA sequencing. The sequence of an operator mutant is also described. The methods are of general use in sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments have shown that this operator contains multiple sites recognized by the lambda phage repressor. We believe we have identified three of these sites.

The nucleotide sequence in the promoter region of the gene N in bacteriophage lambda.

The sequence of 18 nucleotides in the region preceding the initiation of transcription of the gene N of bacteriophage lambda has been determined to be as follows (see article). The basic approach used for the sequence determination involved Escherichia coli DNA polymerase I-catalyzed elongation of the octadecanucleotide primer, dT-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, possessing the appropriate polarity and nucleotide sequence corresponding to the 5' end of the gene N transcript. Following hybridization of the primer to the r-stand of bacteriophage lambda CI85657, sequences of the newly grown ollgonucleotide chains were determined by a) partial exonuclease digestion followed by two-dimensional fingerprinting; b) determination of pyrimidine tracts; and c) nearest neighbor analyses. Primer elongation was carried out in a controlled manner, the size of the newly grown chains being kept short by the following techniques: a) insertion of a ribonucleotide unit as the 3' terminus of the primer; b) use of a limited number of deoxynucleoside 5'-triphosphates in the elongation reaction; and c) enlongation of the primer using all the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 12. Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end.

In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H. G. (1974) J. Biol. Chem. 249, 217) sequence has been synthesized. The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G). All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group. Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase. The DNA duplex was characterized.

Identification of an exposed region of the immunogenic capsid polypeptide VP1 on foot-and-mouth disease virus.

Iodination of intact foot-and-mouth disease virus results in the selective labeling of VP1, substantiating its exposed location on the virion. A comparison of tryptic peptides revealed that a single tyrosine-containing peptide was labeled with iodine on intact or protease-cleaved virus. The labeled peptide from intact and protease-cleaved virus was characterized by molecular weight sizing and sequence analysis. Carboxypeptidase digestion of intact VP1, limited trypsin-cleaved VP1, and VP1 purified from bacterially contaminated tissue cultures yielded carboxyterminal residues of leucine, valine-arginine, and serine-alanine, respectively. The correlation of these findings with previous data on the amino acid sequence derived from nucleotide sequencing of serotypes A12 and O1 of foot-and-mouth disease virus VP1 places the probable exposed antigenic region of VP1 in a serotype-variable region including residues 136 through 144.

The synthesis of a DNA duplex corresponding to the icosanucleotide sequence at the 5' end of messenger RNA from the gene N of bacteriophage lambda.

In connection with work on the nucleotide sequence of the promoter for the gene N of bacteriophage lambda as well as a study of the mechanism of transcription, a 20-unit long DNA duplex corresponding to the known sequence at the 5' end of the above gene transcript has been synthesized. For synthesis, the required duplex was divided into the following deoxyribooligonucleotides: a) the dodecanucleotide, d-A-T-C-A-G-C-A-G-G-A-C-G (II); b) the octanucleotide, d-C-A-C-T-G-A-C-C- (IV); c) the hexanucleotide, d-G-C-T-G-A-rU (I); and d) dodecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T (III). All of the four olignucleotides were chemically synthesized and characterized by extensive chromatographic and fingerprinting methods (after labeling the 5' ends with[32P]phosphate group). Longer polynucleotides (an icosa- and an octadecanucleotide) were prepared by polynucleotide ligase-catalyzed joining of segments I and III and by joining segments II and IV. The use of the octadecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, in work on the sequence analysis of the promoter is described in the accompanying paper. The octadecanucleotide and icosanucleotide were hybridized together to give the double-stranded duplex.

Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli.

Antigenic determinants of influenza virus hemagglutinin were expressed in Escherichia coli. DNA coding for presequences of hemagglutinin were removed and an ATG codon was placed before DNA coding for mature hemagglutinin. A number of expression plasmids were constructed in which various segments of this reconstructed hemagglutinin DNA were fused to DNA coding for bacterial beta-galactosidase. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.

Sequence variation in the gene for the immunogenic capsid protein VP1 of foot-and-mouth disease virus type A.

The nucleotide sequences have been determined and compared from cloned cDNA genes coding for the foot-and-mouth disease virus (FMDV) immunogenic capsid protein, VP1, from eight different A subtypes: A5 Westerwald/58, A12 119ab (large plaque variant), A22 550 USSR/65, A24 Cruzeiro Brazil/55, A27 Cundinamarca Colombia/76, A32 Venezuela/70, A Venceslau Brazil/76, and A Argentina/79. We have also found sequence variations among different cDNA clones of the A5 and A24 subtypes. There are regions of nucleotide sequence within the VP1 gene that vary considerably among the subtypes as well as other regions that remain relatively constant. One highly variable region (codons 130-171) encodes amino acids previously identified as being exposed on the virus surface and constituting an important immunogenic site of the virus. There potentially exist secondary structures within the viral RNA sequences that code for this immunogenic site that could decrease the fidelity of replication at this sequence. The rapid generation of FMDV variants encouraged by such structures in the RNA could work together with various selective pressures to explain the observed accumulation of immunologically distinct viruses of the FMDV A type.

Gibbon ape leukemia virus-Hall's Island: new strain of gibbon ape leukemia virus.

Gibbon ape leukemia virus-Hall's Island (GaLV-H), a type C virus related to previous isolates of GaLV and simian sarcoma virus, was isolated from a gibbon ape with lymphocytic leukemia from a small colony of free-ranging gibbon apes on Hall's Island near Bermuda. We show here by molecular hybridization experiments that GaLV-H is approximately 60% related to three previous isolates of GaLV (GaLV-SF, GaLV-SEATO, and GaLV-Br) and is less closely related to simian sarcoma virus. The oligopyrimidine pattern of a transcript of the terminal 135 +/- 5 nucleotides of the viral RNA of GaLV-H is similar to that of GALV-Br but distinct from that of GaLV-SF and simian sarcoma virus. GaLV-H thus represents a fifth distinct strain of the infectious primate type C viruses, which among the previously described isolates of GaLV is most closely related to GaLV-Br.

Expression in Escherichia coli of chemically synthesized genes for human insulin.

Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli beta-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from beta-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.

Expression of hepatitis B virus surface antigen in yeast.

The structural gene of Hepatitis B virus surface protein (HBsAg) was introduced into a plasmid capable of autonomous replication and selection in both the yeast Saccharomyces cerevisiae and E. coli. In this plasmid transcription of the HBsAg is initiated by the 5'-flanking sequence of the yeast 3-phosphoglycerate kinase (PGK) gene and terminated by the 3'-flanking region of the yeast TRP1 gene. Yeast cells containing this plasmid produce a new major species of mRNA of 1200 nucleotides in length coding for HBsAg. Viral surface antigen is made in nonglycosylated form at a level of about 1-2 percent of total yeast protein. A small fraction of this polypeptide (2-5 percent) is found in aggregated form upon yeast cell disruption by glass beads. This material is similar in size, density, and shape to the 22nm particle, isolated from the plasma of human hepatitis carriers, and induced comparable levels of HBsAg antibodies in mice when compared with the natural particle.