1. Field of the Invention
The present invention is broadly concerned with novel, low cost vaccine preparations, methods of preparing such vaccines and uses thereof. More particularly, the invention is concerned with vaccines and methods wherein the vaccines comprise recombinantly modified and killed microorganisms including therein recombinant DNA encoding at least one protective protein (e.g., an antigenic protein) and which has been expressed by the microorganism prior to killing thereof. These killed recombinant microorganisms can be directly administered as effective vaccines without the necessity of separation of the expressed protective protein(s) from the microorganisms, which has heretofore been considered essential.
2. Description of the Prior Art
A vast array of vaccines have been developed in the past to provide varying degrees of immunity against diseases. Generally speaking, prior vaccines have been in the form of preparations of dead or attenuated pathogenic microorganisms or antigenic substances extracted from them. In the case of bacterial vaccines, it has been known to genetically engineer bacteria to enhance their value as vaccines. Recombinant DNA techniques can also be used to generate attenuated strains, by deletion of pathogenesis-causing genes, or by engineering the protective epitope from a pathogen into a safe bacterium. It is also common to produce antigens or other protective proteins using conventional recombinant DNA techniques, wherein a plasmid or other appropriate vector is inserted into a bacterial host (e.g., E. coli) which then expresses the desired protein. While such engineered proteins can be effective biopharmaceutical vaccines, it has heretofore been thought essential that the expressed proteins be fully separated from the host recombinant microorganism(s) as a part of vaccine production. However, it is sometimes difficult and time consuming to perform such protein separations, and this significantly increases vaccine costs.
Bordetella bronchiseptica is a respiratory tract pathogen of dogs, pigs, cats, laboratory animals and humans. B. bronchiseptica can cause canine respiratory disease in the absence of prior or concurrent viral respiratory tract infection. Clinically, dogs with bordetellosis (“kennel cough”) exhibit a soft, dry to severe paroxysmal cough and can develop extensive histopathological lesions including edema of the bronchial and retropharyngeal lymph nodes, marked polymorphonuclear infiltration of the respiratory tract mucosa and epithelial necrosis. Canine bordetellosis is remarkably similar to pertussis (whooping cough) caused by Bordetella pertussis infection of humans in terms of clinical disease, pathology and epidemiology. See, Keil, Canine Bordetellosis: Improving Vaccine Efficiency Using Genetic and Antigenic Characterization of Bordetella Bronchiseptica Isolates from Dogs (1999).
Kennel cough affects dogs of all ages, has a worldwide distribution, and can have an incidence as high as 50-90% in facilities housing large numbers of dogs. Outbreaks of kennel cough in vaccinated racing greyhounds and other dogs indicate that the disease continues to be a significant problem and that better vaccines are needed. Indeed, outbreaks in well-vaccinated dogs at racing tracks and kennels result in significant economic losses to the greyhound racing industry and at the very least are a periodic nuisance to dog owners, kennel managers and track administrators.
Current vaccines to prevent kennel cough include low-virulent live strains, whole-cell bacterins and undefined antigenic extracts, which are administered by various routes including parenterally and intranasally. Concerns about the efficacy and safety of current kennel cough vaccines have spurred the development of multivalent, acellular vaccines to prevent the disease. However, present-day vaccines do not provide sufficient disease control.
Filamentous hemagglutinin (FHA) is a secreted (but membrane associated) protein conserved within the genus Bordetella (Leininger et al. Inhibition of Bordetella pertussis Filamentous Hemagglutining-mediated Cell Adherence with Monoclonal Antibodies. FEMS Microbiology Letters 1993; 106:31-8.). The structural gene for the FHA of B. pertussis (fhaB) has been cloned and sequenced (Relman et al., Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci USA April 1989; 86(8):2637-41). FHA is essential for bacterial adherence to eukaryotic cells (Relman et al., Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci USA April 1989; 86(8):2637-41). Additionally, the immunologic response against FHA is protective in animal models of infection with B. pertussis (Locht et al. The Filamentous Hemagglutining, a Multifaceted Adhesin Produced by Virulent Bordetella. Supplemental Molecular Microbiology 1993; 9:653-60.; Brennan M J, and Shahin S. Pertussis. Antigens That Abrogate Bacterial Adherence and Elicit Immunity. American Journal of Respiratory Critical Care Medicine 1996; 154:S145-S149.).
While the protective benefits of FHA have been recognized for some time, the immunodominant regions have only recently been identified. Using a panel of monoclonal antibodies, Leininger et al. found two immunodominant domains (type I domain located near the COOH-terminus, type II domain located near the NH2-terminus) within the FHA protein (Leininger et al. Immunodominant Domain Present on the Bordetella pertussis Vaccine Component Filamentous Hemagglutining. Journal of Infectious Disease 1997; 175:1423-31.). Pepscan analysis, using monoclonal antibodies that recognized the type I immunodominant domain, indicated that the epitope for these antibodies was within the amino acid sequence RGHTLESAEGRKIFG (SEQ ID No. 1). Finally, convalescent whooping cough serum, as well as post vaccination serum, contained antibodies that specifically recognize the type I region of FHA.
In order to further characterize the antigenic makeup of the FHA of B. pertussis, Wilson et al. characterized polyclonal anti-FHA reactive clones identified in a phage display library (Wilson et al. Antigenic Analysis of Bordetella pertussis Filamentous Hemagglutining with Phage Display Libraries and Rabbit Anti-filamentous Hemagglutining Polyclonal Antibodies. Infectious Immunology 1998; 66:4884-94.). They determined that the portion of FHA between residues 1929-2019 contained the most immunodominant linear epitope of FHA. They also concluded that because this region contains a factor X homologue (Sandros and Tuomanen. Attachment factors of Bordetella pertussis: mimicry of eukaryotic cell recognition molecules. Trends Microbiol, August 1993; 1(5): 192-6.) and the type I domain peptide defined by Leininger et al. (RGHTLESAEGRKIFG) (SEQ ID No. 2) peptides derived from this region are strong candidates for future protection studies.
Pertactin is the other protein used by B. bronchiseptica to adhere to the respiratory tract. Pertactin gets its name from the fact that it is the only protein that is capable, by itself, of inducing protective immunity against disease. Variations in nucleotide sequence, predicted amino acid sequence, and size of the pertactin proteins expressed in canine B. bronchiseptica isolates have been identified and have been confirmed from researchers working with swine strains of B. bronchiseptica as well as with strains of B. pertussis isolated from whooping cough cases. It is clear that canine vaccine strains of B. bronchiseptica and field isolates from vaccinated dogs with kennel cough do not express the same types of pertactin protein.