Bordetella bronchiseptica vaccine

Details Bordetella bronchiseptica vaccine Bordetella bronchiseptica vaccine

1999-03-30

2001-09-04

Nguyen, Dave Trong (Department: 1633)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S240100, C424S254100, C514S002600, C435S069100, C435S069700, C530S350000

Reexamination Certificate

active

06284256

ABSTRACT:

The present invention is concerned with a vaccine protective against
Bordetella bronchiseptica
infection in dogs, with polypeptides characteristic of
B. bronchiseptica
and with the preparation thereof using rec. DNA-technology.
The genus Bordetella is comprised of four species:
Bordetella pertussis
(
B. pertussis
),
Bordetella parapertussis
(
B. parapertussis
),
Bordetella bronchiseptica
(
B. bronchiseptica
) and
Bordetella avium
(
B. avium
). Bordetella are small gram-negative coccobacilli, obligate aerobic, often bipolar stained, and cytochrome oxidase positive. Colonies on Bordet-Gengou medium are surrounded by a zone of hemolysis except for
B. avium.
All species of Bordetella cause very similar disease with respect to adherence, proliferation, clinical symptoms, and histopathology. Infants of man and animals are most susceptible to infection with Bordetella. Here the disease is most severe and mortality is the highest.
B. bronchiseptica
is primarily a pathogen of laboratory, domestic and wild animals and only occasionally man. Rabbits, guinea pigs, rats, non-human primates, dogs, swine, cats, horses and foxes are often infected in epidemic.
B. bronchiseptica
most notably causes kennel cough in dogs and atrophic rhinitis in piglets. In dogs the infectious process is largely limited to the tracheobronchial tree and is characterized by proliferation on the tracheal epithelium, after adherence to the cilia. The most severe symptoms of the disease are excessive tracheal mucus accumulation, vomiting, pulmonary lesions and weight loss. Dogs have a dry, harsh hacking cough. Infection of piglets with
B. bronchiseptica
is characterized by turbinate atrophy, snout deformity, pneumonia and reduced weight gain. Although it seemed clearly established that
B. bronchiseptica
was the responsible agent of atrophic rhinitis, considerable evidence now indicates that
Pasteurella multocida
is the major phathogen with
B. bronchiseptica
possibly playing an inducing or opportunistic role. The reported carrier states, without clinical signs, are high for dogs, swine and rabbits.
Several virulence factors have been identified in Bordetellae. These include: pertussis toxin, filamentous hemagglutinin, fimbriae, adenylate cyclase, dermonecrotic toxin, tracheal toxin, and hemolysin. These virulence factors are not expressed in all species, for example the gene encoding pertussis toxin in
B. pertussis
is present as a silent gene of the chromosome of
B. parapertussis
and
B. bronchiseptica
. Besides these virulence factors there are probably more, yet unidentified, factors involved in pathogenicity of the bacteria.
Bordetella infections start at the ciliated cells of the respiratory tract. Bacterial adherence is a prerequisite for the initiation of infection since otherwise the flushing action of the cilia removes the bacteria together with other particles from the trachea. Adherence of Bordetella to ciliated cells is mediated by serologically different fimbriae and the filamentous hemagglutinin (FHA) (FHA is not found in
B. avium
however). Fimbriae are hairlike structures, composed of identical subunit proteins, extending from the bacterial cell surface. The FHA is a surface-associated protein, also excreted into the extracellular environment and able to agglutinate a variety of erythrocytes. Both fimbriae and FHA expression is regulated by the bvg-locus although expression of fimbrial subunit genes, in
B. pertussis
, is also influenced by the length of a stretch of 13-15 cytosine residues in front of the fimbrial subunit genes. All virulent Bordetellae express adherence factors on their surface whereas nonvirulent strains do not. Since these adhesins are essential for the initiation of the disease, these are attractive vaccine components.
The purpose of the present invention is to provide a recombinant-DNA vaccine against
B. bronchiseptica
infection. Research has been focused on adhesion factors since prevention of adhesion, as a result of an immune response directed against adhesion factors, will prevent an infection. In
B. bronchiseptica
several serological fimbriae and FHA are responsible for adherence of the bacterium to the ciliated tracheal epithelial cells. When the host has an immune response against the adhesion factors of the microbial organism no colonization will be possible. The bacteria will be killed before any toxins are produced and no clinical symptoms will develop.
A vaccine according to the invention, preventing colonization of
B. bronchiseptica
, preferably contains as much as possible of the components necessary for adherence. A recombinant vaccine can be constructed as a subunit vaccine. It is however not known how many serologically different fimbriae are produced and what the contribution in adherence of each factor (fimbriae and FHA) is. For vaccine development it is necessary to investigate the contribution to adherence of all components separately. The proteins of interest can be used as a subunit vaccine after overproduction in a suited microbial organism.
According to the present invention three different subunit genes coding for adherence factors of
B. bronchiseptica
are isolated and characterized.
Accordingly, the present invention is concerned with a substantially pure preparation of
B. bronchiseptica
fimbrial protein or polypeptide fimbrial protein fragments (fimbrial polypeptides) having at least part of one of the amino acid sequences selected from SEQUENCE ID NO's: 2, 4 or 6.
Furthermore, the present invention is not only concerned with the fimbrial protein and polypeptides but also with DNA of SEQUENCE ID NO's: 1-3 and fragments thereof, and with polynucleotides which hybridize to said DNA and fragments thereof and which code for a polypeptide having the properties of the fimbrial protein of
B. bronchiseptica.
The present invention is concerned also with a polynucleotide which codes for a polypeptide having the immunogenic properties of the fimbrial protein of
B. bronchiseptica
wherein at least part of the codons of the DNA of SEQUENCE ID NO's: 1, 3 or 5 or of the fragments thereof, or of the abovementioned hybridizing polynucleotide is replaced by alternative codons for the same amino acid.
The fimbrial protein and polypeptides derived thereof are able to elicit an immune response against
B. bronchiseptica.
Small antigens often are useless as immunogens. Therefore the fimbrial protein or polypeptides may be prepared as homopolymers (a multitude of indentical fimbrial polypeptides coupled) or heteropolymers (one or more fimbrial polypeptide coupled to one or more different fimbrial polypeptides, or coupled to one or more different polypeptides characteristic of
B. bronchiseptica
or an other pathogen), or may be coupled to one or more other compounds in order to enhance immunogenicity.
According to the present invention the fimbrial polypeptide, in any of the modifications mentioned above, may be prepared by recombinant DNA techniques or may be prepared synthetically, e.g. by homogeneous or by solid state polypeptide synthesis.
The particular amino acid sequences of suitable immunogenic fimbrial polypeptides may be derived from the amino acid sequence according to SEQUENCE ID NO: 2, 4 or 6 and optionally also from the spatial configuration of the fimbrial protein. A number of methods has been developed to predict the location of immunogenically important epitopes on proteins. The outcome of the combined predictions gives a good forecast of antigenic sites.
Suitable fimbrial polypeptides may be selected from the most hydrophilic parts of the fimbrial protein, e.g. by applying the technique described by Hopp and Woods [T. P. Hopp and K. R. Woods (1981): Proc. Natl. Acad. Sci, USA. 78, 3824-3828]. Another suitable method for selecting such polypeptides is described by Chou and Fasman [P. Y. Chou and G. D. Fasman (1987) Advances in Enzymology 47, 45-148].
Various additional algorithms can be used to finally predict the antigenically important regions at the
B. bronchiseptica
fimbrial p

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