Dennis G. Kleid

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.

Synthesis, cloning, and expression of hormone genes in Escherichia coli.

Publisher Summary This chapter focuses on the synthesis, cloning, and expression of hormone genes in Escherichia coli. The genetically engineered bacteria is a valuable source of peptide hormones. The chapter also focuses on the insulin project and provides an overview of the recombinant DNA and chemical DNA synthesis techniques. Genes for large hormones, proteins, and enzymes can be made by chemical DNA synthesis. However, in most cases it is preferable to isolate the natural DNA sequence by cloning a reverse transcript of the appropriate messenger RNA (mRNA). This approach to gene isolation works well if a method is available for detecting the desired gene sequence among the shotgun library of bacterial or viral clones. Detection of the desired clone usually is difficult except for the most abundant protein, and thus mRNA species. The most important function for synthetic DNA can be in trimming, lengthening, or changing natural sequences to obtain efficient expression of the desired peptide product.

Chapter 22. Vaccine Synthesis by Recombinant DNA Technology

Publisher Summary This chapter discusses the introduction and expansion of new techniques of recombinant DNA or “gene splicing” combined with a resurgence of work on adjuvants. This area has received a great deal of interest from both researchers and commercial enterprises. Work on prevention of infectious diseases continues to be a thriving area of research using the traditional technologies. Vaccination with a live-attenuated vaccine gives a mild or unapparent infection that leads to the induction of virus neutralizing antibodies. These antibodies protect the vaccinated individual from a subsequent infection with the normal pathogen. The protection is generally long lasting and quite effective for many human diseases, such as polio, mumps, measles, rubella, yellow fever. Recently, subunit vaccines or “split virus” vaccines have been investigated that contain only parts of the virus. The most important immunogenic proteins from the virus, the viral surface proteins, are included in these preparations. Many other veterinary vaccines, such as those for Mareks disease, infectious bovine rhinotracheitis, transmissible gastroenteritis, etc., are in common use. The approach that has received the most effort has been the production in the bacterial systems of immunogenic proteins that can induce protective antibodies when incorporated into vaccines. The total nucleic acid sequence of the cloned gene is determined by DNA sequencing methods, and thus by deduction of the amino acid sequence of the surface protein is determined. The gene of interest is then recovered from the E. coli plasmids using restriction endonucleases and reintroduced into other plasmids called expression vectors. An innovative use of recombinant DNA methods has been the creation of novel attenuated viruses or bacteria by the specific removal of genes or gene sequences that cause the virus to be pathogenic. The only example of this has been recently reported concerning the Herpes simplex virus. Advantages to the vaccines derived from recombinant DNA technology are potential for higher safety margin, less expensive products because of high but less costly stablity and quality control process, producing vaccines for diseases not amenable to current technology, possibility of product formulation but not possible with the currently available materials, and possibility to develop the proprietary vaccine products.

STRUCTURE OF THE HEMAGGLUTININ GENE OF H0N1 STRAIN OF HUMAN INFLUENZA VIRUS AND ITS EXPRESSION IN ESCHERICHIA COLI

ABSTRACT The structure of the hemagglutinin gene of the H0N1 strain of human influenza virus has been compared to those of other strains. It was found that the H0 hemagglutinin showed the highest homology to the H2 hemagglutinin, suggesting a close geneological relationship between the two. To express the H0 viral HA in Escherichia coli it was placed under control of the lac operon of E. coli. To achieve a high level of expression it was necessary to fuse the H0 hemagglutinin to the E. coli protein β-galactosidase. Fused proteins containing selected portions of HA were specifically precipitated by anti WSN antiserum and therefore, contained antigenic determinants.