Transformed Pichia expressing the pertactin antigen

Details Transformed Pichia expressing the pertactin antigen Transformed Pichia expressing the pertactin antigen

1995-06-02

2001-03-06

Allen, Marianne P. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S254230

Reexamination Certificate

active

06197548

ABSTRACT:

The present invention relates to the expression of heterologous protein in yeast, more particularly to the production of Bordetella pertactin antigens in Pichia, novel expression vectors containing the DNA sequences, and Pichia strains transformed therewith.
Bordetella pertussis
causes whooping cough, an acute respiratory disease which is serious and debilitating in humans, children being particularly susceptible. The organism is responsible for approximately 1 million deaths each year, although this is being to some extent controlled in the developed countries by large scale immunisation programmes. It has been found hat immunisation against
B. pertussis
is very effective at preventing the disease, and that failure to vaccinate does lead to increased incidence of the disease. In practically all areas, immunisation is effected using a whole cell
B. Pertussis
vaccine which has been found to be relatively effective in preventing the disease and infant mortality.
However, public acceptance of whole cell vaccines has decreased due to side-effects and controversy over rare neurological complications attributed to such vaccine preparations. Consequently, researchers have been looking for safer, effective, acellular vaccines consisting of purified Bordetella antigens.
Surface antigen of
B. pertussis
is known to elicit a humoral and a cellular immune response in humans. It is disclosed as ACAP in European Patent Application published under No. 162639 and is now known as P.69 (I. G. Charles et al. Proc. Natl. Acad. Sci. USA, vol. 80, 3554-3558 (1989)). It is likely to be an important component of any future acellular vaccine against
B. pertussis
infections.
B. parapertussis
and
B. bronchiseptica
are closely related to the
B. pertussis
organism.
B. parapertussis
is also responsible for outbreaks of whooping cough in man (Zeuler et al. J. pediatr. 9:493-497 (1946);
B. bronchiseptica
is known to cause respiratory diseases in animals, particularly atrophic rhinitis in pigs (Harris and Switzer Am. J. Vet. Res. 29 777-785 (1968)).
B. parapertussis
and
B. bronchiseptica
appear to present antigens related to
B. pertussis
P.69. with molecular masses of 70 and 68 kDa respectively. These Bordetella antigens, which are referred to hereinafter as ‘pertactin antigens’, are known to bind to the BB05 antibody but appear to have different immunogenic properties (I. G. Charles et al. Proc. Natl. Acad. Sci. USA Vol. 80 3554-3558 (1989)). Only small amounts of pertactin antigen can be isolated from cultures of Bordetella organism. It is preferable for the production of antigens on a commercial scale, to be able to produce large quantities.
E. coli
is known as a host organism for the manufacture of heterologous proteins such as antigens, in quantity, but has certain drawbacks since it contains toxic pyrogenic factors (lipopolysaccharides from the cell wall) which must be rigorously excluded from the final product. The ease with which these factors may be excluded will depend on the method of purification. However, it would be advantageous to eliminate the possibility of contamination altogether simply by using a non-toxic organism as the host, such as yeast.
When baker's yeast,
Saccharomyces cerevisiae
, is used as the host organism, poor expression levels of heterologous protein are frequently obtained. (Kingsman, et al., Biotechnology & Genet. Engin. Reviews. Vol. 3 377-416, 1985). Use of the yeast
Pichia pastoris
as a host for the expression of heterologous protein is also known (European Patent Publication Nos. 0180899 and 0263311). However, expression of membrane proteins in yeast is generally problematic since these proteins can interact with yeast cell membranes causing toxic effects to the yeast cell and reduced product yields. Examples of such difficulties have been described and include the expression of polyoma virus middle-T antigen (Belsham, et al. Eur. J. Biochem. 156 413-421, 1986); expression of the bacterial membrane protein OmpA (Janowitz, et al. Gene 20, 347-358, 1982); and expression of influenza virus haemagglutinin (Jabbar, et al., Proc. Natl. Acad. Sci. USA 82, 2019-2023, 1985).
The present inventors have found a means of producing good levels of expression of the Bordetella pertactin antigens by culturing Pichia transformants containing at least one copy of the DNA encoding a pertactin antigen or an antigenic fragment thereof.
Accordingly, in a first aspect of the invention there is provided a Pichia microorganism transformed with DNA for the expression of a pertactin antigen whose amino acid sequence is at least 95% homologous with that set forth in
FIGS. 1A
,
1
B or
1
C (SEQ ID NO: 1,2 or 3), or an antigenic fragment thereof.
It is preferable if the amino acid sequence described above is at least 98% homologous with that set forth in
FIGS. 1A
,
1
B or
1
C (SEQ ID NO: 1, 2 or 3) or an antigenic fragment thereof.
A pertactin antigen from
B. pertussis
includes the antigen whose amino acid sequence is at least 95% homologous with, but is preferably substantially the same as, that set out in
FIG. 1A
(SEQ ID NO: 1). This antigen is denoted P.69. A pertactin antigen from
B. bronchiseptica
includes the antigen whose amino acid sequence is at least 95% homologous with, but is preferably substantially the same as, that set out in
FIG. 1B
(SEQ ID NO: 2). This antigen is denoted P.68. A pertactin antigen from
B. parapertussis
includes the antigen whose amino acid sequence at least 95% homologous with, but is preferably substantially the same as, that set out in
FIG. 1C
(SEQ ID NO: 3). This antigen is denoted P.70.
The DNA for the expression of a pertactin antigen may encode a larger precursor which has a molecular weight of approximately 94 kD and which is processed within the cell to the desired antigen. In the case of P.69 the precursor is approximately 93.5 kD. The DNA encoding it has been cloned and sequenced by Charles et al., (PNAS, 86, pp 3554-3558, (1989)). The precursor of the P.68 antigen of
B. bronchiseptica
is approximately 94 kD and the precursor for P.70 of
B. parapertussis
is approximately 95 kD.
Pichia microorganisms transformed with DNA for the expression of an antigenic fragment of a pertactin antigen are also encompassed by the invention. The fragments preferably contain no more than 50 amino acid residues. More preferably they contain between 5 and 25 residues. The fragments most preferably comprise a defined antigenically effective sequence which essentially consists of amino acid residues 547 to 552 of the P.69 protein of
B. pertussis.
This sequence is: PGPQPP (SEQ ID NO: 4)
The corresponding sequence for other strains of
B. pertussis
and for strains of
B. parapertussis
and
B. bronchiseptica
can be readily determined by lining up the amino acid sequence of the P.69 antigen, the P.70 antigen or the P.68 antigen respectively with the P.69 sequence shown by Charles et al (1989) referred to hereinbefore.
The fragments described above also include a sequence which essentially consists of the amino acid residues 544 to 566 of the P.69 protein of
B. pertussis.
This sequence is : APQPGPQPPQPPQPQPEAPAPQP (SEQ ID NO: 5)
This sequence and the corresponding sequence for the P.70 antigen of
B. parapertussis
and the P.68 antigen of
B. bronchiseptica
can be aligned. A further fragment of interest is a 60 kD fragment encoded by the C terminal end of the DNA for P.69 which has been identified by Charles et. al. (1989) referred to hereinbefore as encoding an antigenic fragment of P.69.
Transformation of the organism may be carried out by any known method in the literature (Beggs, Nature 275, 104-109 (1978)). It is preferable to use the sphaeroplast method described by Cregg et al., Bio/Technology 5 479-485 (1987). The Pichia organism is preferably transformed with an expression cassette. Expression cassettes include DNA sequences in addition to that encoding the sequence of interest, in this instance the DNA encoding a pertactin antigen, such as transcriptional and translational initiation and termination sequences. The

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