VACCINE COMPRISING A NON-TOXIC IMMUNOGENIC DERIVATIVE OF CLOSTRIDIUM BOTULINUM TYPE D NEUROTOXIN
The present invention relates to immunogenic derivatives of
Clostridium botulinum neurotoxin, DNA encoding such derivatives,
bacterial expression systems comprising DNA encoding such derivatives,
non-C/ostridium botu/inum-based bacterial expression systems expressing
immunogenic botulinum neurotoxin derivatives, vaccines for combating C.
botulinum neurotoxins, methods for the preparation of immunogenic derivatives of C. botulinum neurotoxin and to methods for the preparation
of vaccines for combating C. botulinum neurotoxin.
Clostridium botulinum is a species of the large bacterial genus
Clostridium. Bacteria belonging to this genus are spore-forming anaerobic
Gram positive bacilli. The species C. botulinum can be sub-divided into types and the different types produce several toxins, e.g. such as types A,
C, D and E. Types C and D are generally pathogenic to cattle, sheep, pigs,
man, goats, donkeys, fish, horses, mules, dogs and birds. Botulinum
neurotoxin (BoNT) causes botulism poisoning which commonly occurs in
cattle and sheep in South Africa, Australia, South America and various
other countries. Pathogenesis or poisoning usually results after the ingestion of carrion or decomposed material contaminated with BoNT
produced by C. botulinum bacteria. Clostridial neurotoxins are one of the
most toxic substances known and usually result in the death of the
affected or poisoned animal. The target sites of the BoNT are the
cholinergic nerve endings of neurons in the animal. The BoNT affects
these nerve endings by acting as a zinc-dependent endoprotease to cleave
polypeptides that are necessary for exocytosis of neurotransmitter
containing vesicles. Cleavage of these polypeptides leads to blockage of
transmitter release which results in paralysis and thereafter usually death of the affected animal. BoNT is a protein with an approximate molecular
mass of 1 50 kDa. BoNT may be viewed as being composed of three
functional domains, i.e. a carboxyl-terminal 50 kDa domain which mediates
binding to the target neurons, a 50 kDa middle domain which assists or is
responsible for internalisation of the BoNT, and an amino-terminal 50 kDa
domain which functions as a zinc protease. Botulism can be prevented by
the use of vacines. Botulism vacines currently available are generally
formalin inactivated culture supernatants of C. botulinium types C and D
grown in a corn steep liquor medium in dialysis tubing. Production of
these vacines suffers from several drawbacks including instability of the
C. botulinium vacine production strains, low levels of toxin production,
damage of antigenic determinants during toxoiding and long production
times. Large amounts of experimental animals are involved in quality
control, which should be reduced if possible for ethical reasons. Working
with highly toxic preparations and processing of the toxin for inactivation
with formalin that can destroy the immunogenicity and protective ability
of the antigen also form part of the current problems associated with
current botulism vaccines.
According to one aspect of the invention, there is provided a non-
toxic immunogenic derivative of C. botulinum type D neurotoxin or an immunogenic fragment thereof, said derivative or fragment carrying at
least one mutation in its amino acid sequence, not found in wild-type D
neurotoxins.
Non-toxic is defined as the intra peritoneal injection of 0.2 ml of the
culture supernatant of a bacterial strain containing immunogenic
derivatives of the C. botulinum type D neurotoxin in adult mice, not
resulting in death for 10 days.
The derivative or immunogenic fragment thereof may carry a
plurality of mutations in it's amino acid sequence, the mutations being
selected from at least one of replacement mutations, substitution mutations, deletion mutations, insertion mutations, and inversion
mutations.
According to another aspect of the invention, there is provided a non-toxic immunogenic derivative of C. botulinum type D BoNT which
comprises a polypeptide having the deduced amino acid sequence of
sequence ID No. 1 or a fragment, analog or derivative thereof.
According to a further aspect of the invention, there is provided a
non-toxic immunogenic derivative of C. botulinum type D BoNT which
comprises a polypeptide having the deduced amino acid sequence of
sequence ID No. 2 (amino acids 1 to 399) or a fragment, analog or
derivative thereof.
More specifically, the invention provides a non-toxic immunogenic
derivative of C. botulinum type D BoNT which comprises a polypeptide
having an amino acid sequence which is at least 75%, more preferably
85%, identical to an amino acid sequence selected from the group
consisting of:
(i) amino acids 887 to 1 285 of sequence ID No. 1 , and
(ii) amino acids 1 to 399 of sequence ID No. 2.
An immunogenic fragment thereof is understood to be a fragment that, although not comprising the full length amino acid sequence of the
derivative of the type D neurotoxin, still comprises regions of the derivative
that are capable of inducing a protective immune response in the host
animals.
A mutation is understood to be a change in the nucleic acid
sequence of the derivative type D BoNT in comparison to the nucleic acid
sequence of the wild-type type D BoNT.
As mentioned above, the mutation may be a replacement,
substitution, deletion, insertion or inversion, or a combination thereof. A
mutation can e.g. be such that one or more amino acids of the type D
BoNT are replaced by other amino acids, with different characteristics.
Accordingly, the invention also relates to derivatives of type D BoNT
according to the invention, wherein at least one mutation is a replacement
mutation and/or wherein at least one mutation is a deletion or insertion.
When two or more mutations are made, combinations of
replacement and deletion/insertion mutations are equally possible.
Non-toxic immunogenic derivatives of type D BoNT according to the invention may be made by introducing mutations in the gene encoding the
type D BoNT. The mutated DNA fragments may then be cloned in a nucleotide sequence, such as a suitable expression plasmid and
subsequently be expressed in a suitable host cell.
According to another embodiment of the invention, there is provided a nucleotide sequence comprising a mutated or recombinant DNA fragment
that has as a characteristic that it encodes a non-toxic immunogenic
derivative of C. botulinum type D BoNT or an immunogenic fragment
thereof according to the invention.
Accordingly, the invention provides a nucleic acid characterised by
the nucleotide sequence of at least one of sequence ID No. 1 and
sequence ID No. 2 (nucleotides 58 to 1 254), or a fragment of said
nucleotide.
In other words, the invention provides a nucleic acid comprising a
nucleotide sequence which encodes a non-toxic immunogenic derivative
of C. botulinum type D BoNT or an immunogenic fragment thereof, said
nucleotide sequence being selected from the group consisting of sequence
ID No. 1 , sequence ID No. 2 (nucleotides 58 to 1 254), and a fragment of
said nucleotide sequence.
More preferably, the invention comprises a nucleotide sequence
comprising a polynucleotide having at least 75%, preferably 85%, identity
to a member selected from the group consisting of:
(i) a polynucleotide of sequence ID No. 1 ;
(ii) the complement of (i);
(iii) a polynucleotide comprising nucleotides 58 to 1 254 of
sequence ID No. 2; and (iv) the complement of (iii),
said polynucleotide encoding a non-toxic immunogenic derivative of C.
botulinum type D BoNT or an immunogenic fragment thereof.
More preferably, the invention comprises a nucleotide sequence
comprising a polynucleotide having at least 75%, preferably 85%, identity
to a member selected from the group consisting of:
(i) a polynucleotide of sequence ID No. 3; and
(ii) the complement of (i),
said polynucleotide encoding a genetically non-toxic immunogenic
derivative of C. botulinum type D BoNT or an immunogenic fragment
thereof.
The polynucleotide may be DNA or RNA. Preferably, the
polynucleotide is plasmid DNA.
A suitable bacterial expression system for expressing the non-toxic immunogenic derivatives of type D BoNT according to the invention are
Gram positive bacteria such as Bacillus brevis, Bacillus subtilus or Gram negative bacteria such as Escherichia coli. It is also envisaged that other
expression systems may be used for expressing non-toxic immunogenic
derivatives of type D BoNT. Other possible expression systems may be a
suitable yeast expression system, for example Pichia pastoris.
In another embodiment of the invention, there is provided a Gram
positive bacterial expression system, comprising a nucleotide sequence
according to the invention encoding a non-toxic immunogenic derivative
of C. botulinum type D BoNT or an immunogenic fragment thereof .
In a further embodiment of the invention, there is provided a Gram
negative bacterial expression system comprising a nucleotide sequence according to the invention encoding a non-toxic immunogenic derivative
of C. botulinum type D BoNT or an immunogenic fragment thereof.
According to a further embodiment of the invention, there is
provided a vaccine for protection against botulism caused by C. botulinum
type D BoNT, said vaccine comprising a non-toxic immunogenic derivative
of C. botulinum type D neurotoxin or an immunogenic fragment thereof
according to the invention, and a physiologically acceptable carrier.
Such vaccines may be made by admixing an immunologically sufficient amount of a non-toxic immunogenic derivative or derivatives of
C. botulinum type D BoNT according to the invention and a physiologically
acceptable carrier.
An immunologically sufficient amount is understood to be the
amount of non-toxic immunogenic C. botulinum type D BoNT derivative
that is capable of inducing a protective immune response in a host animal.
Thus still another embodiment of the invention relates to vaccines for protection against botulinum caused by C. botulinum type D BoNT that
comprises a non-toxic immunogenic derivative of C. botulinum type D
BoNT according to the invention or an immunogenic fragment thereof, and
a physiologically acceptable carrier.
Optionally, one or more compounds having adjuvant activity may be
added to the vaccine, e.g. Alhydrogel, Alum or Saponin.
The vaccine may be administered to all hosts sensitive to C.
botulinum type D BoNT, such as cattle, mules, sheep, goats, horses, birds, humans, etc.
It will be appreciated that an effective dosage of 1 ml
subcutaneously for sheep and goats or 2 ml subcutaneously for cattle,
mules and horses of the vaccine will be administered per animal. The
dosage is administered once and may be repeated after a month or two.
According to another aspect of the invention, there is provided a
method for preparing a non-toxic immunogenic derivative of C. botulinum
type D BoNT or an immunogenic fragment thereof according to the
invention, which method comprises expressing in a suitable host cell, a
nucleotide sequence according to the invention.
Non-toxic immunogenic derivatives of C. botulinum type D BoNT
according to the invention may be made by replacing or modifying amino
acids of the polypeptide or protein. The non-toxic immunogenic
derivatives may also be prepared by introducing mutations in the gene or
nucleic acid sequence encoding the C. botulinum type D BoNT. It will be
appreciated that the non-toxic immunogenic derivatives may be prepared
by suitable recombinant DNA techniques known in the art. The
recombinant DNA or fragments thereof may then be cloned in a nucleotide
sequence, such as a suitable expression vector, and subsequently be
expressed.
According to a further aspect of the invention, there is provided a
method for the preparation of a vaccine for combating C. botulinum type
D BoNT poisoning, which method comprises admixing a non-toxic
immunogenic derivative of C. botulinum type D BoNT according to the
invention and a physiologically acceptable carrier.
The vaccine may be administered by a variety of suitable routes
including internal, for example oral, nasal, or parenteral administration, for
example by the intravenous, subcutaneous and intramuscular.
According to a further aspect of the invention, there is provided a
vector which contains a polynucleotide of sequence ID No. 1 which
encodes a polypeptide having the deduced amino acid sequence of
sequence ID No. 1 .
According to another aspect of the invention, there is provided a
vector which contains a polynucleotide of sequence ID No. 2 (nucleotides
58 to 1 254) which encodes a polypeptide having the deduced amino acid
sequence of sequence ID No. 2 (amino acids 1 to 399).
Accordingly, the invention provides a vector which includes a
nucleotide sequence according to the invention which encodes a non-toxic
immunogenic derivative of C. botulinum type D BoNT or an immunogenic fragment thereof.
The invention also extends to a host cell genetically engineered with
a vector as described herein.
The host cell may be any suitable host cell as a yeast or bacterium.
Accordingly the host cell may be B. subtilus, E. coli. or B. brevis, e.g. B.
brevis strain 47.
The vector may be any suitable vector known in the art, such as a suitable plasmid.
The invention also extends to a method for preparing a non-toxic
immunogenic derivative of C. botulinum type D BoNT as herein described,
which method comprises expressing in a Gram positive bacterium, a
nucleotide sequence according to the invention encoding a non-toxic
immunogenic derivative of C. botulinum type D BoNT.
The invention also provides a method for preparing a non-toxic
immunogenic derivative of C. botulinum type D BoNT as herein described,
which method comprises expressing in a Gram negative bacterium, a nucleotide sequence according to the invention encoding a non-toxic
immunogenic derivative of C. botulinum type D BoNT.
The nucleotide sequence may be a polynucleotide of sequence ID
No. 1 or sequence ID No. 2 (nucleotides 58 to 1 254).
The nucleotide sequence may be expressed under the control of its
native promoter or under the control of a heterologous promoter.
The Gram positive bacterium may be selected from the group consisting of Bacillus brevis and Bacillus subtilus.
The Gram negative bacterium may be E. coli.
According to yet another aspect of the invention, there is provided
a process for producing cells capable of expressing a non-toxic
immunogenic derivative of C. botulinum type D BoNT or an immunogenic
fragment thereof, said process comprising genetically engineering cells
with a vector or plasmid as herein described.
According to a further aspect of the invention, there is provided a method of vaccinating an animal against C. botulinum type D BoNT, said
method comprising administering an immunologically effective amount of
a vaccine as herein described to the animal.
According to another aspect of the invention, there is provided a
substance or composition for use in a method of vaccinating an animal against C. botulinum type D BoNT, said substance or composition
comprising a non-toxic immunogenic derivative of C. botulinum type D
BoNT or an immunogenic fragment thereof as herein described, and said
method comprising administering an immunologically effective amount of
said substance or composition to said animal.
According to yet a further aspect of the invention, there is provided
use of a non-toxic immunogenic derivative of C. botulinum type D BoNT
or an immunogenic fragment thereof as herein described in the
manufacture of a vaccine to vaccinate animals against C. botulinum type
D BoNT poisoning.
The invention will now be described, by way of non-limiting
example, with reference to the following Examples and Figures in which:
Figure 1 shows the structure of a suitable expression-secretion
vector. The closed bar indicates the 5 region of the MWP gene containing
multiple promoters and a signal peptide-coding sequence. The open bar
indicates a multiple cloning site (MCS). The DNA and amino acid
sequences of these regions are shown in the upper part of the Figure.
Vertical arrows along the top of the DNA sequence indicate transcription
start sites 1 -5. SD 1 and SD2 are the ribosome-binding sites located
upstream of the dual translation initiation sites. The signal peptide-coding
sequence is underlined (reference 7) . The broken line indicates the
position of the nucleic acid sequence encoding a non-toxic immunogenic
derivative of C. botulinum type D BoNT;
Figure 2 is a genetic map of a gene in accordance with the invention
incorporated in a suitable vector for maintenance in E. Coli;
Figure 3 is a genetic map indicating sequencing coverage and
sequence primer location. Included is a list of primers used and their
sequences;
Figure 4 is an analysis of recombinant plasmids through comparative
restriction enzyme digests. Lane 1 represents DNA molecular weight
marker standards. Lanes 2 and 4 represent plasmid extractions from B.
brevis transformants compared with lane 6, representing a PNU 21 1
plasmid profile. Hind III and Pst I restriction enzyme digests of PNU 21 1 recombinant plasmids (lanes 3 and 5) were compared with the same
restriction enzyme digests of plasmid PNU 21 1 before ligation with the
gene fragment in accordance with the invention and GeneOp-ABC plasmid
carrying the gene fragment according to the invention (lanes 7 and 8
respectively). The arrows indicate the respective sizes of the DNA
fragments in base pairs (bp) . The arrows indicating the DNA fragments of 1 207 bp, represent the genes according to the invention as digested from
the recombinant PNU 21 1 plasmids and the GeneOp-ABC plasmid;
Figure 5 is a PAGE electrophoretogram of B. brevis culture supernatant demonstrating secretion of recombinant heterologous COOH-
heavy chain fragment of the C. botulinum type D neurotoxin into modified
PY medium by B. brevis 1 1 5 as determined by western blot analysis (Fig
1 3) . Lane 1 represents the protein profile of the culture supernatant of a
B. brevis strain transformed with PNU 21 1 without the gene fragment
according to the invention. Protein profiles, of samples of culture
supernatant of B. brevis 1 1 5 (transformed with PNU 21 1 ligated with the
gene according to the invention) prepared under non-reducing and reducing
conditions, are presented in lanes 2 and 3 respectively. The arrow
indicates the position of the recombinant protein;
Figure 6 is a Western blot analysis of a PAGE protein profile of B.
brevis culture supernatants demonstrating secretion of recombinant heterologous COOH-heavy chain fragment into modified PY-medium by B
brevis 1 1 5. The recombinant fragment protein band was detected by
treating the filter with polyclonal antibodies raised against a crude extract
of the native neurotoxin of C. botulinum type D. Lane 1 represents the
protein profile of a crude extract of the native neurotoxin protein complex
of C. botulinum type D. Protein profiles of samples of culture supernatant
of B. brevis 1 1 5, prepared under non-reducing and reducing conditions, are
presented in lanes 2 and 3, respectively. Lane 4 represents the protein
profile of the culture supernatant of a B. brevis strain transformed with
PNU 21 1 without the gene fragment according to the invention. The arrow indicates the position of the recombinant fragment protein according
to the invention; and
Figure 7 is a growth curve of a fermentation culture of B. brevis
strain 1 1 5 in modified PY-medium.
Sequence ID No. 1 is a nucleotide sequence of a synthetic gene
encoding a non-toxic immunogenic derivative of C. botulinum t pe D toxin
(Sequence ID No. 1 -nucleotide and amino acid sequences).
Sequence ID No. 2 shows a nucleic acid sequence of a gene
encoding a non-toxic immunogenic derivative of type D BoNT according to
the invention (sequence ID No. 2 - nucleotide and amino acid sequences)
including portions of a suitable plasmid which are immediately upstream
and downstream of the gene. Included is a list of restriction sites and
amino acid composition.
Example
A novel nucleotide sequence or gene according to the invention encoding a non-toxic immunogenic derivative of C. botulinum type D toxin
(BoNT) was created using an amino acid sequence of C. botulinum type D
strain South Africa (Dsa) (Reference 3) to start from. The nucleotide
sequence of amino acids Nos. 887 - 1 285 of the heavy chain COOH(Hc)
neurotoxin fragment was recorded (Sequence ID Nos 1 and 2) . The
nucleotide sequence or gene was redesigned to have the optimal codons
for expression in B. brevis by making use of the B. brevis codon table.
Materials and methods
Bacterial strains and vectors.
An Escherichia coli strain JM 109 (Reference 3) was used for the
amplification of the gene according to the invention. Bacillus brevis strain
47-5Q (JMC no. 8970) was obtained from the Japanese Collection of
Microorganisms, The Institute of Physical and Chemical Research, Wako-
shi, Saitama, Japan. Plasmid PNU 21 1 was obtained from S Udaka, Department of Applied Biological Sciences, Nagoya University, Japan..
Clones were selected by growth on LB agar plates supplemented with 50
/g/ml ampicillin. Positive clones were cultured in LB-broth supplemented
with 50 μ g/ml ampicillin (Reference 5). Plasmid extractions were
performed according to the method of Reference 1 9. Plasmids were
digested with restriction enzymes Pst I and Hind III and analyzed on a 1 %
agarose gel to confirm the presence of the gene fragment according to the
invention (Reference 1 9). The gene fragment according to the invention
was digested with Pst I and Hind III, and the gene fragment ligated with
plasmid PNU 21 1 (S Udaka, Department of Applied Biological Sciences,
Nagoya University, Japan - Reference 27). B. brevis strain 47 - 5q
(deposited under No. JMC 8970 Japan Collection of Microorganisms, The
Institute of Physical and Chemical Research, Waka-shi, Saitama, Japan,
350-01 ) was cultured in T2U-medium (Udaka and Yamagata 1 993 -
reference 28). Transformations were performed according to the method
of Reference 2. The B. 6τe s transformants were cultured in T2U-medium
supplemented with 10 //g/ml erythromycin and plasmid extractions performed according to the method of Reference 1 . Plasmids were
analyzed for the correct insertion by restriction enzyme Pst I and Hind III digests and analyzed on a 1 % agarose gel. Transformants were screened
for expression in T2U-medium, PM-medium (Reference 28), 5YC-medium
(Reference 2) and PY-medium (Reference 1 5) through colony blot analysis
using polyclonal antibodies raised against the native neurotoxin of C. botulinum type D.
Enzymes and reagents.
Calibration Proteins for SDS gel electrophoresis were from Boehringer
Mannheim. ECL Western blotting detection reagents and ECL protein
molecular weight markers were obtained from Amersham International.
Shrimp alkaline phosphatase was obtained from Boehringer Mannheim. T4
DNA Ligase and restriction enzymes were obtained from Promega. HRP
conjugated Protein G was obtained from Zymed Laboratories Inc. Hybond-
C nitrocellulose membranes, supplied by Amersham International, were
used for colony blot analysis. PVDP membranes used for Western blots
were obtained from Microcept. Onderstepoort Biological Products (OBP)
Onderstepoort, South Africa supplied polyclonal antibodies against the
native neurotoxin of C. botulinum type D. Standardized (u/ml) C. botulinum
type D neurotoxin was also obtained from OBP, Onderstepoort, South
Africa. DAB substrate kit was obtained from Zymed Laboratories Inc. Difco
supplied Proteose Peptone. Fluka was the supplier of Polyethylene glycol
6000.
Synthetic gene or nucleotide sequence in accordance with the
invention.
A synthetic gene or nucleotide sequence according to the invention was
created using the amino acid sequence of C. botulinum type D strain South Africa (Dsa) Reference 14 as a starting point. The nucleotide sequence of
amino acids nos. 887 to 1 285 of the heavy chain COOH (He) neurotoxin
segment was recoded. The gene according to the invention was designed
to include optimal codons for expression in Bacillus brevis by making use
of the Bacillus brevis codon table. A codon optimization program was used
for construction, eliminating any rare (4 to 8 in a thousand) to very rare (4
or less in a thousand) codons and then to use representative numbers of the remaining codons. Restriction enzyme recognition sites for Pst I and
Hind III were included for cloning purposes.
The gene was synthesized and cloned into a suitable vector
[GeneOp vector (Operon technologies)] as shown in Figures 2 and 3. The
vector carrying the synthetic gene fragment according to the invention
was transformed into an Escherichia coli strain Jm 109 according to the
method of Reference 1 9.
Antisera.
Antiserum used for immunoblots was prepared by hyper-immunization of horses against C. botulinum serotype D neurotoxin (OBP). Antibodies
present in the serum, cross-reacting with Bacillus brevis cell proteins, were removed through adsorption with B. brevis acetone powder (Reference 5).
Bacillus brevis recombinants.
As mentioned above, the GeneOp vector carrying the synthetic gene
fragment (GeneOp-ABC) was transformed into Escherichia coli strain JM
109 (Reference 1 9). Clones were selected by growth on LB agar plates
supplemented with 50 / g/ml ampicillin. Positive clones were cultured in
LB-broth supplemented with 50 //g/ml ampicillin (Reference 1 9). Plasmid
extractions were performed according to the method of Reference 1 9.
( 1 989) . Plasmids were digested with restriction enzymes Pst I and Hind III
and analyzed on a 1 % agarose gel to confirm the presence of the gene
fragment (Reference 1 9). All restriction enzyme digests were performed
according to the instructions of the manufacturer. Bacillus brevis strain 47- 5Q (JMC no. 8970) was transformed with PNU 21 1 according to the
method of Reference 28. Clones containing PNU 21 1 were selected by
growth on T2U plates supplemented with 10 /g/ml erythromycin
(Reference 28). B. brevis containing the PNU 21 1 plasmid was cultured in
T2U-medium supplemented with 1 0 //g/ml erythromycin. Plasmids were
extracted according to the method of Reference 2 and analyzed on a 1 %
agarose gel. GeneOp-ABC was digested with Pst I and Hind III, and
dephosphorilated using Shrimp alkaline phosphatase according to the instructions of the manufacturer. The GeneOp-ABC preparation (5 //g) was
ligated with 5 μg PNU 21 1 , digested with Pst I and Hind III, using T4 DNA ligase according to the instructions of the manufacturer. Transformation
of 5. brevis with the ligation mixture was performed according to the method of Reference 28. Control transformations with undigested PNU
21 1 were included. Clones were selected by growth on T2U plates
supplemented with 10 //g/ml erythromycin. PNU 21 1 transformants with
the gene fragment insert were screened by immuno colony blot analysis.
Identified transformants that produced a heterologous protein were picked
from the plates and grown on fresh plates. The B. brevis transformants
were cultured in T2U-medium supplemented with 10 //g/ml erythromycin
and plasmid extractions performed according to the method of Reference
2. Recombinant plasmids were analyzed for the correct insert by restriction enzyme Pst I and Hind III digests in comparison with Pst I and Hind III
digested GeneOP-ABC plasmid material on a 1 % agarose gel.
Immuno colony analysis.
Plates of transformants were covered with sterile nitrocellulose membranes
for 1 to 3 hours. After removal the colonies transferred to the membranes
were lysed by treating the membranes with lysing buffer (50 mM Glucose,
10 mM EDTA, 25 mM Tris, 0.5 % lysozyme, pH 8.0) for 1 hr at 37 °C.
Membranes were transferred in a Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-Rad) with colony side facing for 30 min at 25 V (Trans-
Blot SD Semi-Dry Electrophoretic Transfer Cell Intruction Manual). After transfer, the membranes were washed 2 x for 1 5 min in TBS buffer
(Reference 1 9) at room temperature. The membranes were then incubated
in TBS containing 1 0 //g/ml Dnase I at room temperature for at least 10
min with gentle shaking on an orbital shaker. The membranes were placed
in TBS buffer supplemented with 1 0 % fat free milk powder for 1 hr or
overnight at 4 °C. Adsorbed Onderstepoort C. botulinum antiserum type D ( 1000 units/ml) was diluted 1 : 200 in TBS ( 1 % milk powder) and used
as primary antibody solution. Membranes were incubated with primary
antibody solution for 1 h at room temperature on an orbital shaker. The
membranes were then washed 3 times for 5 min at room temperature. Finally, the membranes were incubated for 1 hr in Horseradish peroxidase
(HRP) conjugated Protein G (Zymed), diluted 1 : 3000 in TBS (1 % milk
powder), and afterwards washed 3 times for 5 min in TBS. A positive
reaction was detected by using a 3,3 = -Diaminobenzidine
tetrahydrochloride (DAB) substrate kit (Zymed) for HRP according to the
instructions of the manufacturer.
Preservation of cultures.
B. brevis strains were cultured in T2U medium at 37 °C for 24 h. Cultures
were stored in T2U medium containing 25 % (v/v) glycerol at -70 °C.
Protein expression and secretion.
B. brevis transformants carrying the gene fragment according to the invention were cultured in PY medium (Reference 1 5), modified as follows:
[Proteose peptone (20g), yeast extract (1 .5g), glucose ( 1 5g), and uracil (0.5g)/liter] supplemented with mineral mixture (0.01 835% FeSO4.7H2O,
0.0008 % MnSO4.7H2O, 0.0001 % ZnSO4.7H2O), 0.1 % MgSO4.7H2O and 20 //g/ml erythromycin. Modified PY medium ( 100 ml) was inoculated
with 1 ml of a glycerol store culture and incubated for 4 days at 30 °C as
a shake culture. Culture were harvested and centrifuged to remove
bacterial cells. Protein profiles of the culture supernatant were prepared
and compared with the negative control strain (B. brevis transformed with
PNU 21 1 ) through SDS-PAGE electrophoresis, PAGE electrophoresis and
Western blot analysis.
PAGE electrophoresis. PAGE electrophoresis (Bio-Rad Mini Protean II) on a 5% gel were
performed as described by Hames and Rickwood ( 1 990) using the non-
dissociating high pH discontinuous system. Sample preparation occurred
under both reducing and non-reducing conditions. Samples were diluted
1 :4 with stacking gel buffer supplemented with 40 % sucrose and 0.008 % Bromophenol blue. To create reducing conditions when necessary,
samples were diluted 1 :4 with stacking gel buffer supplemented with 40
% sucrose, 0.008 % Bromophenol blue, 8 % SDS and 2 % Dithiothreitol
and boiled for 5 min. Gels were fixed for 20 min in fixing solution (400 ml
ethanol, 1 00 ml glacial acetic acid, 500 ml distilled water) and then
exposed to destain solution (300 ml ethanol, 80 ml glacial acetic acid, 620
ml distilled water) for 2 min. Gels were stained for 10 min in staining
solution (0.1 % Coomassie blue in destain solution) and then destained in
destain solution.
Western blot analysis Proteins were transferred from PAGE gels to PVDF membranes using the
Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BioRad Instruction
manual) . Towbin transfer buffer (BioRad Instruction manual) was modified
by increasing the methanol concentration in the buffer from 200 ml to 220
ml. The Western blot analysis was completed following the method as
described for the Immuno colony analysis. Onderstepoort C. botulinum
antiserum type D ( 1000 units/ml) was, after adsorption, diluted 1 :200 and
used as primary antibody. Horseradish peroxidase (HRP) conjugated Protein G was diluted 1 : 3000 and used as secondary antibody. The ECL Western
blotting detection reagent kit (Amersham International) was used following
the Instruction Manual of the manufactures of the ECL kit. Positive reactions were visualized using the Lumi-lmager (Boehringer Mannheim)
and Lumi Analyst Image Analysis software program (Boehringer Mannheim) .
The concentration of the recombinant fragment protein band, identified by
Western blot analysis, was calculated according to the relative % of the
band to the total protein concentration loaded per sample using the
LumiAnalyst Image Analysis software program (Boehringer Mannheim).
Fermenter production A non-toxic neurotoxin fragment according to the invention was produced
in a B. Braun Biotech International fermenter in 10 liter modified PY-
medium supplemented with 0.05 % Tween 20. T2U medium ( 100 ml) was
inoculated with 1 ml of a glycerol store culture and incubated for 24 h at
37 °C as a shake culture. The 24-h T2U culture was then used to
inoculate each of 5 flasks containing 100 ml modified PY-medium with 20-
ml culture. The modified PY shake cultures were incubated for 24 hr at 37
°C and then used as inoculum for the 1 0 liter quantity fermenter system.
After completion of the fermentor run at day 4, the contents were
harvested and centrifuged to remove the bacterial cells. The culture supernatant was analysed for the presence of the protein fragment
according to the invention with dot-blot hybridization based on the method
as described for the Immuno colony assay. Samples were also withdrawn
at daily intervals during the fermenter run and culture supernatant analyzed
with dot-blot hybridization to monitor the protein expression and secretion
of the protein fragment during the process.
Vaccination of animals.
The culture supernatant was mixed in a 1 : 1 ratio with 50 % Aluminium
Hydroxide adjuvant (OBP) and used as a vaccine to immunize groups of 5
healthy outbred BalbC (of the colony maintained at OBP) mice, each
weighing 1 8 g to 20 g. The mice were each injected subcutaneously with 0.2 ml. After 21 days, two groups of 5 immunized mice and 1 0 control mice each, were challenged with OBP C. botulinum type D standard
neurotoxin. One group of immunized mice and one group control mice
were injected intraperitoneally into each of the mice with 0. 2 ml of toxin diluted in saline to give a final concentration of 0.05 U. The other group
of immunized and control mice were each challenged with 0.03 U toxin.
The mice were observed for 24 h for survival, signs of botulism or death.
RESULTS AND DISCUSSION
Synthetic gene composition.
The nucleotide sequence of the synthetic gene fragment according to the
invention with restriction enzyme sites is shown in Figures 3 and Sequence
ID Nos 1 and 2. The size of the gene is 1 207 bp and the resulting protein
after expression 399 amino acids long. The percentage of rare codons
used was 0.3 %. The %GC content is 34.6 %, compared to the %GC of
B. brevis of 42.7 to 47 % (Reference 27) and to that of C. botulinum of
26 to 28 %.
Bacillus brevis recombinants.
The construction of the PNU 21 1 vector is shown in Fig. 1 . At the Pst I
site, the gene fragment according to the invention was fused to the 5 =
region of the MWP gene. Restriction enzyme digests of the GeneOp vector
carrying the gene fragment before and after amplification in E. coli, control
plasmid PNU 21 1 and recombinant PNU21 1 plasmids isolated from the
transformants were compared (Fig 4). A 1 .2 Kbp fragment could be excised from the plasmids with Pst I and Hind III. This demonstrated the
presence of an insert in the transformants with a size comparable with that
of the synthetic gene according to the invention.
Protein expression and secretion.
Immunoreactive transformant colonies were identified. One of these
transformants (B. brevis 1 1 5) was cultured on modified PY-medium and
the culture supernatant subjected to PAGE electrophoresis and Western
blot analysis to demonstrate protein expression and secretion. As
expressed in B. brevis, the gene according to the invention produces and
secretes a polypeptide into the culture medium of modified PY-medium (Fig. 5) that reacts in Western blots (Fig. 6) with antisera to serotype D
botulinum toxin.
Fermenter production
Process constraints due to genetic instability of genetically manipulated or
altered microorganism is more significant on commercial scale than
laboratory scale. Production started with a batch-growth phase in the
production medium, used as a 20 % inoculum for 10 liter medium in the
fermenter. Figure 7 represents the growth curve of a typical fermenter run.
Temperature was maintained at 30 °C throughout the run. The pH values
were monitored and used as a growth indicator. Bacterial growth reached
the stationary phase in approximately 4 days.
The fermenter was set at maximum value for oxygen saturation. As
bacterial growth entered the exponential growth phase the PO2 value
decreased dramatically and only started to recover to the initial value as
the growth reached the stationary phase. This demonstrates the
dependency on oxygen for maximal growth of B. brevis. Maximum values
for production and secretion of the protein according to the invention into
the culture medium were obtained after 4 days when growth entered the
stationary phase. Higher yields of the secreted protein according to the
invention were detected in the culture fluid using the fermenter system,
compared with a batch culture. This might be attributed to more sufficient
continuous supply of oxygen in the fermenter, especially after the
stationary phase of growth is reached and the bacteria starts to shed the
cell wall proteins into the medium, than in a batch culture. Extracellular
production of 1 g/liter of the protein according to the invention was
obtained using the fermenter system.
Vaccination of animals.
To obtain an indication of the protective antigenicity of the protein or
polypeptide according to the invention in the formulation of a vaccine
against C. botulinum type D neurotoxin, a crude culture supernatant was
used to vaccinate mice. Mice challenge studies with 0.03 U and 0.05 U toxin, 21 days after vaccination, resulted in death of all the control mice
in 24 h. In contrast, 60 % and 20 % mice vaccinated with the culture
supernatant still survived for at least 24 h post challenging with 0.03 U
and 0.05 U, respectively. The culture supernantant appeared to have no
detrimental effects on the mice.
Advantages of the invention are that it provides a relatively simple,
relatively inexpensive method of producing non-toxic immunogenic
derivatives of C. botulinum type D BoNT and vaccines for combating C.
botulinum type D BoNT. This method of vaccine production would involve fermenter technology, a unique method for botulism type D vaccine
production compared to the current analysis production method. The vaccines can be produced or manufactured relatively quickly and the
vaccine is non-toxic perse. Since the product of the fermentation process
is non-toxic, the production procedures result in little or no health risk to
production personnel.
References:
The following references are incorporated herein.
1 . Clayton, M. A., Clayton, J. M., Brown, D.R. and Middlebrook, J.L.
( 1 995). Protective vaccination with a recombinant fragment of
Clostridium botulinum neurotoxin serotype A expressed from a
synthetic gene in Escherichia coli. Infect. Immun. Vol. 63, p. 2738-
2742.
2. Hardy, K. G. ( 1 985). Bacillus Cloning Methods, p. 1 B 1 7. In: DNA
Cloning Volume II, a practical approach. (Ed.) D.M. Glover. IRL
Press, Oxford, Washington DC.
3. Hanahan, D. ( 1 985) . Techniques for Transformation of E. coli. p. 109 B 1 1 1 . In: DNA Cloning Volume I, a practical approach. (Ed.)
D.M. Glover. IRL Press, Oxford, Washington DC.
4. Hames, B.D. and Rickwood, D. ( 1 990). Gel Electrophoresis of
Proteins A Practical Approach. Second Edition. IRL Press at Oxford
University Press, Oxford.
5. Harlow, E. and Lane, D. ( 1 988). Antibodies A Laboratory Manual.
Cold Spring Harbor Laboratory Press.
6. Hatheway, CH. ( 1 990) . Toxigenic Clostridia. Clin. Microbiol. Rev.
Vol. 3. P. 66-98.
7. Ichikawa, Y., Yamagata, H., Tochikubo, K. and Udaka, S. ( 1 993) . Very efficient extracellular production of cholera toxin B subunit
using Bacillus brevis. FEMS. Microbiol. Lett. Vol. 1 1 1 . p. 21 9-224.
8. Jansen, B.C., Knoetze, P.C. and Visser, F. ( 1 976) The antibody
response of cattle to Clostridium botulinum type C and D toxoids.
Ondertepoort J. Vet. Res. Vol. 43. p.1 65-1 74.
9. Kiyatkin, N., Maksymowych, A. B. and Simpson, L. L. ( 1 997) .
Induction of an immune response by oral administration of recombinant botulinum toxin. Infect. Immun. Vol.65. p. 4586-4591 .
1 0. Konishi, H., Sato, T., Yamagata, H. and Udaka, S. ( 1 990). Efficient
production of human σ-amylase by a Bacillus brevis mutant. Appl.
Microbiol Biotechnol. Vol. 34, p. 297-302.
1 1 . Lamanna, C. ( 1 959) The most poisonous poison. Science, Vol. 1 30,
p. 736-772.
1 2. LaPenotiere, H.F., Clayton, M.A. and Middlebrook, J.L. ( 1 995).
Expression of a large nontoxic fragment of botulinum neurotoxin
serotype A and its use as an immunogen. Toxicon, Vol. 33, p.
1 383-1 386.
1 3. Makoff, A. J., Ballantine, P.S., Smallwood, A. E. and Fairweather,
N.F. ( 1 989). Expression of tetanus toxin fragment C in E. coli: its
purification and potential use as a vaccine. Biotechnology Vol. 7. p.
1043-1046. 14. Moriishi, K., Koura, M., Fujii, N., Fujinaga, Y., Inoue, K., Syuto, B.
and Oguma, K. ( 1 996) . Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin
types C1 and D, and PCR detection of this gene from Clostridium
botulinum type C organisms. Appl. Environ. Microbiol. Vol. 62. p.
662-667.
5. Nagahama, M., Michiue, K. and Sakurai, J. ( 1 996). Production and
purification of Clostridium perfringens alpha-toxin using a protein-
hyper producing strain, Bacillus brevis 47. FEMS Microbiol. Lett.
Vol. 145. p. 239-243.
6. Potter, K.J., Bevins, A.M., Vassilieva, E.V., Chiruvolu, V.R., Smith,
T., Smith, L.A. and Meagher, M.M. ( 1 998). Production and
purification of the heavy-chain fragment C of botulinum neurotoxin,
serotype B, expressed in the methylotrophic yeast Pichia pastoris.
Protein Expr. Purif. Vol. 1 3, p. 357-365. 7. Romanos, M. A., Makoff, A.J., Fairweather, N. F., Beesley, K. M.,
Slater, D. E., Rayment, F. B., Payne, M. M. and Clare, J. J. (1 991 ). Expression of tetanus toxin fragment C in yeast: gene synthesis is
required to eliminate fortuitous polyadenylation sites in AT-rich
DNA. Nucleic Acid Res. Vol. 1 9. p. 1461 -1467.
8. Rosenberg, J.S., Middlebrook, J.L. and Atassi, M., Z. ( 1 997) .
Localization of the regions on the C-terminal domain of the heavy
chain of botulinum toxin A recognized by T lymphocytes and by
antibodies after immunization of mice with pentavalent toxoid.
Immunological Investigations, Vol 26, p. 491 -504.
19. Sambrook, J., Fritsc. E. F. and Maniatis, T. (1989). Molecular
Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor
Laboratory Press.
20. Shuler, L.S. and Kargi, F. (1992). Bioprocess Engineering Basic
Concepts. (Eds.) M. Hays, R. Maes, and W. Thomas. Prentice Hall,
Englewood Cliffs, New Jersey, U.S.A.
21. Smith, L. DS. (1977). Botulism, The Organism, Its Toxins, The Disease. (Ed.) A. Barlows, Charles C Thomas Publusher, Springfield,
Ilinois, U.S.A. 22. Smith, L. A. (1998). Development of recombinant vaccines for
botulinum neurotoxin. Toxicon, Vol.36, p. 1539-1548.
23. Sterne, M. and Wentzel, L.M. (1950). A new method for the large-
scale production of high-titre botulinum formol-toxoid types C and
D. J. Immun. Vol.65. p. 175-183.
24. Sugiyama, H. (1980). Clostridium botulinum Neurotoxin.
Microbiological Reviews. Vol.44, p.419-448.
25. Takagi, H.A., Miyauchi, A., Kadowaki, K. and Udaka, S. (1989).
Potential use of Bacillus brevis HPD31 for the production of foreign
proteins. Agric. Biol. Chem. Vol.53, p.2279-2280. 26. Udaka, S. (1990). Potential use of Bacillus brevis for enzyme
production. Ann. N. Y. Acad. Sci. Vol.613. p.582-583.
27. Udaka, S., Tsukagoshi, N. and Yamagata, H. (1989). Bacillus brevis,
a host bacterium for efficient extracellular production of useful
proteins. Biotechnol. Genet. Eng. Rev. Vol.7. p. 113-146.
28. Udaka, S. and Yamagata, H. (1993). High level Secretion of
heterologous proteins by Bacillus brevis. Methods Enzymol. Vol.
217. p.23- 33.