Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1998-04-17
2002-12-31
Graser, Jennifer E. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S185100, C424S190100, C424S193100, C424S203100, C424S234100, C530S350000
Reexamination Certificate
active
06500435
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the veterinary field. More specifically, the invention relates to the prophylaxis and therapy of pleuropneumonia in pigs.
BACKGROUND OF THE INVENTION
Pleuropneumonia is a major respiratory disease in pigs and causes severe economic losses in pig farming in many countries including the United States and Canada. The disease is caused by the bacterium
Actinobacillus pleuropneumoniae
(previously also referred to as
Haemophilus pleuropneumoniae
) and is considered to be one of the most important disorders of the bronchial tubes in pigs. Frequently, the disease is fatal.
Actinobacillus pleuropneumoniae
is known to exist in twelve infective serotypes.
Since pleuropneumonia can be induced by inoculating pigs with sterile culture supernatants of
A. pleuropneumoniae
, extracellular toxic proteins are assumed to be involved in the development of the pneumonic lesions. There is growing evidence that qualitative or quantitative differences in toxic activities exist between the twelve serotypes of
A. pleuropreumoniae
. Hemolytic and cytotoxic activities have been reviewed by T. A. Bertam,
Can. J. Vet. Res
. 54: S53-S56 (1990). Two different hemolytic activities were reported by Frey and Nicolet,
J. Clin. Microbiol
. 28: 232-236 (1990), whereas four antigenically different activities were distinguished by Kamp and Van Leengoed,
J. Clin. Microbiol
. 27: 1187-1191 (1989). Whether such activities are functions of one or more molecules is not known.
Vaccines proposed thus far for preventing infections by
Actinobacillus pleuropneumoniae
are mostly based on whole live cells, attenuated cells, lysates, culture supernatants, or extracts of
A. pleuropneumoniae
. WO-A-80,02113 (or Canadian Patent 1,189,790) teaches a vaccine for controlling pleuropneumonia in pigs, containing
A. pleuropneumoniae
cells, cell fragments etc. and, as an adjuvant, material derived from
Bordetella pertussis
. EP-A-420,743 proposes a vaccine containing inactivated toxin of serotype 1 and optionally an inactivated toxin of another serotype of
A. pleuropneumoniae
; it provides protection against serotype 1 and partial protection against other serotypes. EP-A-354,628 discloses a universal vaccine against
A. pleuropneumoniae
, which contains extracellular proteins from two different serotypes, and is effective against all
A. pleuropneumoniae
serotypes. Although these known vaccines provide protection against some or even all of the field strains of
A. pleuropneumoniae
, the active compounds are not known. As a result, control, verification, and standardisation of vaccines is difficult, since the ratio between active components cannot be optimized and inactive and sometimes adverse components are always present in the vaccines.
SUMMARY OF THE INVENTION
The present invention, in one aspect, provides a vaccine for the prevention and/or the treatment of infection by
Actinobacillus pleuropneumoniae
containing at least an immunogenic part of at least one polypeptide selected from the group consisting of cytolytic proteins of
A. pleuropneumoniae
produced by recombinant DNA technology, and detoxified derivatives thereof.
It has been found according to the invention that
Actinobacillus pleuropneumoniae
produces three hemolytic and/or cytotoxic proteins (toxins), hereinafter referred to as cytolytic proteins: Cytolysins I, II and III (ClyI, ClyII and ClyIII). Where the term “Cytolysin” (Cly) is used in the present specification, this shall thus be understood to comprise any extracellular protein produced by any strain of
A. pleuropneumoniae
and producing any adverse effect (be it hemolytic, cytotoxic or other or both) on cells or tissues of an infected animal; where appropriate it shall be understood also to comprise immunogenically active parts of these proteins or derivatives thereof having diminished adverse effects. Protection against infections by any of the known serotypes of
A. pleuropneumoniae
is conferred to an animal by administering an effective amount of all three cytolysins, and partial or complete protection against specific serotypes is conferred by administering one or two of the cytolysins, depending on the serotype or serotypes in question.
Thus, the vaccine of the invention contains at least one of the three cytolysins I, II and III, preferably two, and more preferably three. The cytolysins may be present in the vaccine as the naturally occurring proteins, or they may be present as derivatives containing at least an immunogenic part of the proteins, or as a detoxified equivalent. Detoxification shall be understood to mean that the toxic activity of the proteins has been removed to a sufficient degree or for a sufficient number of the protein molecules to provide a vaccine which does not produce an unacceptable toxic reaction in the producing host and/or in the vaccinated animal, whereas it provides a sufficient immune response. Detoxification can be brought about by chemical, physical or enzymatic treatment of the proteins or by substitution, insertion or deletion of one or more nucleotides in the cytolysin genes resulting in the substitution, insertion or deletion of one or more amino acids in the protein. Detoxification can also be achieved by expression of the toxin gene in the absence of the activator gene.
It was found that the cytolysins are encoded by operons wherein the structural toxin gene is flanked at the 5′ end by a gene encoding a peptide required to activate the toxin, hereinafter referred to as the activator protein. The cytolysins may be present in the vaccine in the activated or non-activated form.
The cytolysins or their derivatives present in the vaccine are preferably obtained by expression of recombinant DNA encoding the proteins mentioned above. The detoxified cytolysins constitute a further embodiment of the present invention.
In another aspect of the invention a process for producing a cytolytic protein of
Actinobacillus pleuropneumoniae
or an immunogenic and/or detoxified derivative thereof is provided, which process comprises the steps of:
a) selecting at least one nucleotide sequence coding for at least an immunogenic part of said cytolytic protein (toxin) optionally including an activator protein, or a derivative thereof;
b) inserting the nucleotide sequence(s) selected in step a) in a vector or an expression vector;
c) transforming a host cell, preferably a host cell that is capable of secreting said cytolytic protein, with the vector obtained in step b);
d) cultivating the host cell of step c) to express the nucleotide sequence(s) of step a),
e) recovering and optionally purifying the protein from the culture;
f) optionally modifying the protein to produce a detoxified protein.
In yet another aspect, the invention is concerned with a process of producing a vaccine wherein at least one, and preferably two, and more preferably three, of the cytolysins or immunogenic parts thereof, thus produced, are combined with an immunologically acceptable carrier and optionally a suitable adjuvant.
The host cell referred to in the process of producing the cytolysins or their derivatives may be a microorganism, preferably a non-pathogenic microorganism capable of expressing at least one nucleotide sequence encoding the cytolysins by having a strong promoter inducing high expression levels or by allowing the introduction of an exogenous promoter system to induce such high expression levels. A suitable host cell is
Escherichia coli.
In a further aspect, the invention provides a nucleotide sequence encoding at least an immunogenic part of a polypeptide selected from cytolytic proteins of
Actinobacillus pleuropneumoniae
optionally including activator proteins and transport proteins, the latter ones being proteins that assist in the secretion of the cytolytic proteins to the periplasma or the medium. The invention also relates to a system that expresses and secretes said nucleotide sequence and to a vector containing at least one of said nucleotide sequences each one preferably operatively linked to a promoter and op
Kamp Elbarte Margriet
Smits Marinus Adrianus
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