U.S. patent application number 11/651346 was filed with the patent office on 2010-10-07 for non-toxic double mutant forms of pertussis toxin as adjuvants.
This patent application is currently assigned to UCB Pharma Limited. Invention is credited to Mark Roberts.
Application Number | 20100255033 11/651346 |
Document ID | / |
Family ID | 26303633 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100255033 |
Kind Code |
A1 |
Roberts; Mark |
October 7, 2010 |
Non-toxic double mutant forms of pertussis toxin as adjuvants
Abstract
The invention relates to the use of an antigen which is a
non-toxic double mutant form of pertussis toxin for the manufacture
of a vaccine composition for intranasal administration to induce an
immune response against B. pertussis infection. The invention also
relates to the use of a non-toxic double mutant form of pertussis
toxin for the manufacture of an adjuvant composition for
stimulating or enhancing a protective immune response of an antigen
co-administered therewith. The non-toxic double mutant is
preferably one in which the glutamic acid 129 amino acid in the
S.sub.1 sub-unit has been substituted by glycine and the arginine 9
amino acid has been substituted by lysine.
Inventors: |
Roberts; Mark; (Glasgow,
GB) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
UCB Pharma Limited
Slough
GB
|
Family ID: |
26303633 |
Appl. No.: |
11/651346 |
Filed: |
January 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09775909 |
Feb 2, 2001 |
7169399 |
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11651346 |
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09179272 |
Oct 27, 1998 |
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09775909 |
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08619600 |
Apr 1, 1996 |
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PCT/GB94/02152 |
Apr 10, 1994 |
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09179272 |
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Current U.S.
Class: |
424/240.1 |
Current CPC
Class: |
C07K 14/235 20130101;
A61P 11/14 20180101; A61P 31/04 20180101; A61K 39/00 20130101 |
Class at
Publication: |
424/240.1 |
International
Class: |
A61K 39/10 20060101
A61K039/10; A61P 31/04 20060101 A61P031/04; A61P 11/14 20060101
A61P011/14 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 1993 |
GB |
9324743.5 |
Oct 5, 1993 |
GB |
9320454.3 |
Claims
1.-36. (canceled)
37. A method of stimulating or enhancing a protective immune
response to an antigen in a mammal, which method comprises
administering to said mammal with the antigen an effective adjuvant
amount of a non-toxic double mutant form of pertussis toxin, said
antigen being one which elicits a protective immune response when
administered with said effective adjuvant amount of said non-toxic
double mutant form of pertussis toxin, wherein said non-toxic
double mutant form of pertussis toxin comprises an S.sub.1 sub-unit
containing an amino acid at position 129 which is other than
glutamic acid and containing an amino acid at position 9 which is
other than arginine.
38. A method according to claim 37, wherein the amino acid at
position 129 in the S.sub.1 sub-unit is glycine.
39. A method according to claim 37 wherein the amino acid at
position 9 in the S.sub.1 sub-unit is lysine.
40. A method according to claim 37 wherein the antigen and the
non-toxic double mutant form of pertussis toxin are administered
simultaneously or sequentially.
41. A method according to claim 40 wherein the antigen and the
non-toxic double mutant form of pertussis toxin are present in
admixture in a composition administered to the mammal.
42. A method according to claim 37 wherein the antigen is selected
from the group consisting of tetanus toxin C-fragment, and one or
more immunogenic fragments thereof.
43. A method according to claim 37 wherein the antigen is selected
from the group consisting of FHA and P69.
44. A method according to claim 43 wherein both FHA and P69 are
administered with the non-toxic double mutant form of pertussis
toxin.
45. A vaccine composition comprising an antigen, an adjuvant
capable of enhancing the immune response to the antigen in a mammal
to which the composition is administered, and a pharmaceutically
acceptable carrier, wherein the adjuvant is a non-toxic double
mutant form of pertussis toxin comprises an S.sub.1 sub-unit
containing an amino acid at position 129 which is other than
glutamic acid and containing an amino acid at position 9 which is
other than arginine.
46. A vaccine composition according to claim 45, wherein the amino
acid at position 129 in the S.sub.1 sub-unit is glycine.
47. A vaccine composition according to claim 45, wherein the amino
acid at position 9 in the S.sub.1 sub-unit is lysine.
48. A vaccine composition according to claim 45, wherein the
antigen is selected from the group consisting of tetanus toxin
C-fragment, and one or more immunogenic fragments thereof.
49. A vaccine composition according to claim 45, wherein the
antigen is selected from the group consisting of FHA and P69.
50. A vaccine composition according to claim 49, which comprises
both FHA and P69.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/775,909, filed Feb. 2, 2001 and now pending, which is a
continuation of U.S. application Ser. No. 09/179,272, filed Oct.
27, 1998, now abandoned, which is a continuation of U.S.
application Ser. No. 08/619,600, now abandoned, which was a
national phase filing under 35 U.S.C. .sctn.371 of PCT
international application no. PCT/GB1994/002152, which was
published under PCT Article 21(2) in English.
FIELD OF THE INVENTION
[0002] This invention relates to vaccine compositions for delivery
to mucosal surfaces, adjuvant compositions for stimulating or
enhancing the protective immunogenic effects of an antigen
co-administered therewith; methods of inducing an immune response
to an antigen or mixture of antigens and methods of stimulating or
enhancing the protective immunogenic effect of an antigen by
co-administering therewith an adjuvant composition.
[0003] More particularly, the invention relates to the mucosal
immunogenic and adjuvant properties of a mutant form of pertussis
toxin.
[0004] The majority of pathogenic microorganisms initiate infection
by attaching themselves to the mucosal epithelial cells lining the
gastro-intestinal, oropharyngeal, respiratory or genito-urinary
tracts. Some pathogens, such as influenza virus, Bordetella
pertussis, or Vibrio cholerae, may remain at or within the mucosal
tissue, while others, such as Salmonella typhi or hepatitis A
virus, possess mechanisms that allow them to penetrate into deeper
tissues and spread systemically. The specific and nonspecific
defence mechanisms of the mucous membranes provide first line
protection against both types of pathogen. Non-specific effectors
include resident macrophages, antimicrobial peptides, lactoferrin
and lysozyme, extremes of pH, bile acids, digestive enzymes, mucus,
shedding of epithelial cells, flushing mechanisms (peristalsis,
ciliary beating, micturation, etc) and competition from local
flora. However, successful pathogens have generally evolved means
to survive the non-specific defences present at the site they
infect and it is the secretory immune system which plays a major
role in protecting against diseases caused by a number of bacterial
and viral pathogens, and is probably the major effector against
pathogens that are restricted to mucosal surfaces. For organisms
that spread systemically, both local and systemic immune responses
are probably needed for optimum immunity.
[0005] A means of stimulating the local and systemic lymphoid
tissues is needed for effective immunisation against many diseases.
Unfortunately, current parenteral immunisation regimes often
stimulate only weak or undetectable secretory responses in the
mucosa. In order to achieve efficient stimulation of the
mucosa-associated lymphoid tissue (MALT), the immunogen needs to be
applied topically to the mucosal surface during the course of
vaccination. However, this is not as straightforward as it seems.
Most non-replicating immunogens are poorly immunogenic when
ingested or inhaled, and soluble proteins are particularly
inefficient mucosal immunogens. This is undoubtedly because the
non-specific defences will readily denature, degrade and eliminate
most soluble proteins resulting in the MALT encountering only
minute quantities of such immunogens.
[0006] Administering large repeated doses of a particular protein
may be expected to enhance the immune response. However, the result
of such immunisation is often the induction of a state of
immunological unresponsiveness known as oral tolerance where the
individual responds poorly to subsequent parenteral immunisation
with the same antigen (it would be more accurately labelled mucosal
tolerance because inhalation of large amounts of soluble proteins
also induces this state). The regulatory mechanisms involved in
initiation of oral tolerance are poorly understood but are believed
to have evolved to prevent animals developing inappropriate and
possibly deleterious immune response to environmental and dietary
proteins. One of the major goals of modern vaccinology therefore is
to devise means of eliciting strong mucosal and systemic immune
responses to soluble proteins, but without inducing mucosal
tolerance.
[0007] Some microbial components such as cholera toxin (CT) or E.
coli heat-labile toxin (LT) or the non-toxic binding portions of
these toxins (CT-B and LT-B) have been found to be potent mucosal
immunogens eliciting strong secretary and circulating antibodies,
and cholera toxin is understood to be the most potent mucosal
immunogen known. However, the reason why such molecules are good
mucosal immunogens has not yet been fully elucidated. One property
that may be important is the ability of these molecules to bind to
mucosal epithelial cells via certain surface receptors, although it
has been found in studies by others that there is not necessarily a
correlation between the ability of an antigen to bind to eucaryotic
cells and its mucosal immunogenicity.
[0008] Thus, as far as we are aware, there is currently no way of
predicting with any certainty whether a given antigen will possess
good mucosal immunogenicity.
[0009] Cholera toxin (CT) is not only immunogenic when administered
mucosally, but is also a mucosal adjuvant that greatly enhances the
responses to co-administered antigens. In mice, only minute
quantities of CT are necessary for the adjuvant effect.
Unfortunately, a dose of 2 .mu.g of CT fed to human volunteers is
diarrheaogenic and a dose of 5 .mu.g induces purging
indistinguishable from classical cholera. Active CT is therefore
clearly unacceptable for administration to humans.
[0010] CT is a bipartite toxin consisting of a protomer (CTA) and a
B pentamer (CTB). CTA is the enzymatic moiety of CT responsible for
the covalent modification of host G proteins and the consequent
toxicity of CT. CTB, which mediates the binding of CT to its
receptor (ganglioside GM.sub.1) on the surface of eucaryotic cells,
is non toxic and is also a good mucosal immunogen. CTB has been
investigated as a mucosal adjuvant by many groups with conflicting
results. CTB obtained from commercial suppliers is prepared from CT
and often contains trace quantities of CT which could be
responsible for the adjuvanticity of CTB reported by some authors.
Furthermore, it has been found that recombinant CTB and the highly
related E. coli heat labile toxin B pentamer (LTB), although
immunogenic themselves, are devoid of adjuvanticity. Thus, Holmgren
et al (Vaccine, Vol II, pp 1179-1184, 1993) have reported that when
highly purified or recombinant CTB was used, they were consistently
unable to observe an adjuvant action of CTB for other antigens
admixed therewith. Holmgren et al concluded that the whole CT
molecule is required for the adjuvant action. They also tested a
mutant form of E. coli heat labile toxin (LT) in which the B
sub-unit was identical to that of the normal B sub-unit, and the A
subunit was identical with the exception of a single amino acid
substitution in position 112 (Glu.fwdarw.Lys). However, the mutant
form which did not possess ADP-ribosylating activity and did not
cause fluid secretion in rabbit ligated loops, failed to give rise
to any significant IgA response against itself and demonstrated no
adjuvant properties.
[0011] Pertussis toxin (PTX), like CT, has an AB.sub.5 structure.
Both CT and PTX are ADP-ribosylating toxins but they have different
cellular receptors and substrates. PTX can produce a myriad of
biological effects and is one of the major protective antigens of
B. pertussis. Inactivated forms of PTX constitute the basis of
current and experimental acellular pertussis vaccines.
[0012] Pertussis toxin (PTX) has been reported to have adjuvant
properties, but a major drawback, in addition to its inherent toxic
properties, is its property of stimulating and enhancing IgE
production thereby leading to anaphylaxis to co-administered
proteins--see for example Mu et al, Infection and Immunity, pp.
2834-2840, July 1993.
[0013] Thus, there remains a need for a mucosal adjuvant which
lacks the toxic and undesirable side effects described above.
DESCRIPTION OF THE RELATED ART
[0014] It has now been found that a particular mutant form of
pertussis toxin is not only lacking in the toxic properties of the
wild type toxin, but has good immunogenic activity when
administered via the intranasal route. Moreover, the said mutant
form of pertussis toxin is also an excellent adjuvant.
[0015] Accordingly, in a first aspect, the invention provides the
use of an antigen which is a non-toxic double mutant form of
pertussis toxin for the manufacture of a vaccine composition for
intranasal administration to induce an immune response against B.
pertussis infection.
[0016] In a second aspect, the invention provides the use of a
non-toxic double mutant form of pertussis toxin for the manufacture
of an adjuvant composition for stimulating or enhancing a
protective immune response of an antigen co-administered
therewith.
[0017] The adjuvant composition is preferably adapted for
administration to a mucosal surface, and in particular for
intranasal administration.
[0018] The non-toxic double mutant form of pertussis toxin is
preferably one in which the glutamic acid 129 amino acid in the
S.sub.1 sub unit has been substituted by another amino acid, such
as glycine.
[0019] It is also preferred that the arginine 9 amino acid has been
substituted by another amino acid for, example by lysine.
[0020] In another aspect, the invention provides a vaccine
composition adapted for intranasal administration, the vaccine
composition comprising a non-toxic double mutant form of pertussis
toxin as hereinbefore defined, and a pharmaceutically acceptable
carrier.
[0021] The said vaccine composition can contain one or more other
pertussis antigens selected from filamentous haemagglutinin (FHA)
and the P69 outer membrane (P69). More particularly the composition
can contain both FHA and P69.
[0022] In a still further aspect, the invention provides a vaccine
composition comprising an antigen and an adjuvant capable of
enhancing the immune response to the antigen in a mammal to which
the composition is administered; characterised in that the adjuvant
is a non-toxic double mutant form of pertussis toxin as
hereinbefore defined.
[0023] A particular vaccine composition is one in which the antigen
is tetanus toxin C fragment.
[0024] The vaccine composition is preferably adapted for
administration to a mucosal surface, and in particular the nasal
mucosa.
[0025] In another aspect, the invention provides a method of
immunising a host such as a mammal (e.g. human) against B.
pertussis infection, which method comprises administering to the
host intranasally an effective amount of a vaccine composition as
hereinbefore defined.
[0026] The invention also provides a method of stimulating or
enhancing an immune response to an antigen in a mammal; which
method comprises co-administering with the antigen an effective
adjuvant amount of a non-toxic double mutant form of pertussis
toxin, as hereinbefore defined.
[0027] In another aspect, the invention provides a vaccine
composition comprising a first antigen and an effective adjuvant
amount of a non-toxic mutant form of pertussis toxin in which the
Glu 129 amino acid in the S.sub.1 sub-unit has been substituted by
another amino acid.
[0028] In another aspect, the invention provides a method of
stimulating or enhancing a protective immune response to an antigen
in a mammal which method comprises co-administering with the
antigen an effective adjuvant amount of a non-toxic mutant form of
pertussis toxin in which the Glu 129 amino acid in the S.sub.1
sub-unit has been substituted by another amino acid, e.g.
glycine.
[0029] Particular examples of non-toxic double mutant pertussis
toxins for use in the present invention are those disclosed in
European Patent Application EP-A-0462534 (Sclavo SpA). A preferred
non-toxic double mutant toxin is the mutant described in Example 1
of EP-A-0462534, in which the arginine 9 residue has been
substituted by lysine, and the glutamic acid 129 residue has been
substituted by glycine. This mutant is referred to hereinafter as
PT 9K/129G.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The vaccine and adjuvant compositions of the invention
typically are formulated as an aqueous solution for administration
as an aerosol or nasal drops, or as a dry powder, e.g. for
inhalation.
[0031] Compositions for administration as nasal drops may contain
one or more excipients of the type usually included in such
compositions, for example preservatives, viscosity adjusting
agents, tonicity adjusting agents, buffering agents and the like.
The antigen or mixture of antigens typically is selected such that
it is non-toxic to a recipient thereof at concentrations employed
to elicit an immune response.
[0032] The pertussis toxin double mutant and a further antigen may
be administered separately, for example, within a short period of
one another, or they may be administered together simultaneously.
When administered together, they may be formulated as a mixture of
discrete entities. Alternatively, they may be chemically linked
together, or may form part of a fusion protein produced by
recombinant DNA methods. The vaccine composition may in addition,
contain one or more further mucosally immunogenically active
antigens.
[0033] In a particular embodiment of the invention, the pertussis
toxin mutant may be combined with one or more other pertussis
antigens, for example filamentous haemagglutinin (FHA), and/or the
69 kilodalton outer membrane protein (P69--also known as pertactin)
from B. pertussis.
[0034] A further example of an antigen that may be co-administered
with the mutant pertussis toxin is the C fragment of tetanus toxin
(hereinafter referred to as Frg C). It has been found that the
immunogenicity of Frg C is markedly enhanced when it is
co-administered with the mutant pertussis toxin.
[0035] The P.69 outer membrane protein of B. pertussis is a protein
of approximately 61 KD molecular weight; see A. J. Makoff et al,
"Protective surface antigen P.69 of Bordetella pertussis: its
characteristics and very high level expression in Escherichia
coli," Bio-Technology, 8, 1030 (1990).
[0036] It can be prepared and isolated according to the method
disclosed in P. Novotny et al: The Journal of Infectious Diseases,
164, 114 (1991), or recombinant material prepared from E. coli by
the method given in the article by A. J. Makoff et al referred to
above. It can bind to eukaryotic cells.
[0037] Purified B. pertussis filamentous haemagglutinin usually
contains polypeptides of differing molecular weight ranging from
98-220 KD, and can be isolated and purified from cell culture
supernatants of B. pertussis, for example as described in the
article by P. Novotny et al referred to above. The filamentous
haemagglutinin is able to bind to eukaryotic cells and cause
haemagglutination of sheep erythrocytes.
[0038] The antigenic molecules of the present invention can be
prepared by isolation and purification from the organisms in which
they occur naturally, or they may be prepared by recombinant
techniques and expressed in a suitable host such as E. coli in
known manner. When prepared by a recombinant method or by
synthesis, one or more insertions, deletions, inversions or
substitutions of the amino acids constituting the peptide may be
made.
[0039] The aforementioned antigens are preferably used in the
substantially pure state. The quantity of the mixture of antigens
administered will depend, in part, upon the purity of the
individual antigens. Thus, for a substantially pure form of the
non-toxic double mutant pertussis toxin, or the P.69 outer membrane
protein, a dose in the range from about 1-100 microgrammes/dose
typically would be administered to a human, the actual amount
depending on the immunogenicity of the preparation in humans when
applied to mucosal surfaces.
[0040] For a substantially pure form of the B. pertussis
filamentous haemagglutinin, a typical dose range would be of the
order given above in respect of the mutant pertussis toxin or P.69
protein. In a typical immunisation regime employing the antigenic
preparations of the present invention, the vaccine may be
administered in several doses (e.g. 1-4), each dose containing
1-100 microgrammes of each antigen. The immunisation regime may
involve immunisation purely by the mucosal route, or a combination
of mucosal and parenteral immunisation. The dosage will in general
depend upon the immunogenicity of the different antigens when
applied to the respiratory tract of animals.
[0041] The invention will now be illustrated, but not limited, by
reference to the examples set forth below, and accompanying Figures
in which:--
[0042] FIG. 1 illustrates the effects of intranasal and
subcutaneous immunisation with PT 9K/129G on bacterial levels in
the lungs of BALB/c mice following challenge with B. pertussis;
[0043] FIG. 2 shows the results of the analysis of bacterial counts
retrieved from nasal lavage following intranasal and subcutaneous
immunisation with PT 9K/129G and subsequent aerosol challenge with
B. pertussis;
[0044] FIG. 3 illustrates the effect of cholera toxin (CT),
pertussis toxin (PTX) and PT-9K/129G on the serum IgG response to
Fragment C of tetanus toxin;
[0045] FIG. 4 illustrates the serum IgG response to PTX, PT-9K/129G
and CT' in mice immunised intranasally;
[0046] FIG. 5 illustrates the effect of CT, PTX and PT-9K/129G on
the serum IgG response to Fragment C, and the effect of
boosting;
[0047] FIG. 6 illustrates the serum response to PTX, PT-9K/129G and
CT in intranasally immunised mice and the effect of boosting;
[0048] FIG. 7 illustrates the effect of CT, PTX and PT-9K/129G on
the secretory IgA response to Fragment C in the respiratory tract
of NIH:S mice;
[0049] FIG. 8 illustrates the secretory IgA responses to CT, PTX
and PT-9K/129G in the respiratory tract of NIH:S mice;
[0050] FIG. 9 illustrates the serum anti-Frg C response in BALB\c
and NIH:S mice; and
[0051] FIG. 10 illustrates the serum anti-PTX and PT-9K/129G IgG
responses in BALB\c and NIH:S mice.
EXAMPLE 1
Intranasal Immunisation with B. pertussis PT-9K/129G Mutant
[0052] Mice were immunised intranasally three times with B.
pertussis PT-9K/129G mutant obtained from Sclavo (4.4 microgrammes
per dose) or ovalbumin (10 microgrammes per dose) the second and
third doses being administered at 28 days and 150 days
respectively. Elispot analyses (see below) were performed after the
second and third doses to determine the immune response in the
lungs of the mice. The antibody responses in the lungs taken seven
days after the second dose, and 5 days after the third dose are
shown in Table 1 below. From the results it can be seen that the
immune response to intranasally administration of mutant pertussis
toxin PT-9K/129G was significantly better than the response
stimulated by ovalbumin (OVA).
TABLE-US-00001 TABLE 1 Lung ELISPOT - Lung ELISPOT - 2nd dose
(ASC/108 3rd dose (ASC/108 Serum lymphocytes) lymphocytes) Response
IgG IgA IqM IgG IgA IgM PT 9K/129G 2500 2706 <50 39 4423 1231
115 OVA <50 <40 <40 40 49 <49 244
EXAMPLE 2
Intranasal Immunisation with a Combination of FHA, P69, and
Pertussis Toxin Mutant PT 9K/129G
[0053] BALB/c mice were immunised intranasally with 10 microgrammes
of each of the above antigens, 36 and 14 days prior to aerosol
challenge with B. pertussis BBC 26. The outer membrane protein of
B. pertussis, P69, was synthesised intracellularly in E. coli and
purified as described in A. J. Makoff et al, Bio/Technology 8, 1030
(1990). Filamentous haemagglutinin (FHA) was provided by SKB under
an exchange of reagents agreement. Antigens were diluted in PBS
immediately prior to immunisation.
[0054] Adult (6-8 weeks) mice were anaesthetised with metathane and
the antigen solution was added to the external nares of the mice as
they recovered consciousness. Antigen was taken into the
respiratory tract by inhalation.
[0055] The results obtained from the analysis of colony forming
units of B. pertussis in the lungs are shown in FIG. 1. For
comparison purposes the corresponding figures obtained from
subcutaneous immunisation are also shown. As can be seen,
immunisation via the nasal route gave results broadly equivalent to
those obtained by the subcutaneous route.
[0056] The results of the analysis of the bacterial counts of B.
pertussis retrieved from nasal lavage are shown in FIG. 2. Again,
the figures obtained by subcutaneous administration are shown by
way of comparison. From the figures, it can be seen that
immunisation by the subcutaneous route gave rise to a slightly
greater reduction in bacterial numbers than was obtained by
intranasal administration up to about the 10 day point, but from
about 10 days onwards, the bacterial numbers showed a greater
reduction in animals immunised via the intranasal route.
EXAMPLE 3
Serum and Secretory Immune Responses to Fragment C, PT, PT 9K/129G
and CT in Intranasally Immunised Mice
[0057] Groups of adult female outbred NIH:S mice were immunised
intranasally (ISN) with 10 .mu.g of fragment C (FRG C) alone or
admixed with 5 ug of cholera toxin (CT), active pertussis toxin
(PTX) or PT-9K/129G 25d apart. The cholera toxin was obtained from
Sigma (Dorset, UK); the active pertussis toxin was obtained from
Calbiochem (Nottingham, UK) or NIBSC (Herts, UK); and the mutant
pertussis toxin PT-9K/129G was obtained from IRIS (Siena,
Italy).
[0058] Prior to immunisation, the mice were anaesthetised with
metathane and the antigen was added to the external nares of the
mice as they recovered consciousness. Antigen was taken into the
respiratory tract by inhalation.
[0059] As a comparison, a group of mice was immunised twice
subcutaneously (S\C) with 10 .mu.g of fragment C adsorbed to
aluminium hydroxide gel (alhydrogel). Serum samples were taken 14
days after primary immunisation and serum and nasal wash samples 14
days after the boost. Antibody responses against each of the
components were determined by ELISA.
[0060] The serum anti-Frg C IgG responses following a single
immunisation are depicted in FIG. 3, in which each point on the
graph represents the mean value obtained from 5 mice. Anti-fragment
C antibodies were not detected in mice immunised I\N with Frg C or
Frg C+PTX. In contrast mice receiving Frg C combined with
PT-9K/129G or CT had significant amounts of anti-Frg C antibodies
in their serum. The levels of anti-Frg C antibodies were similar in
the two groups and were somewhat lower than those in parenterally
immunised mice. Mice immunised I\N with Frg C and CT mounted a very
strong serum IgG response to CT (titres greater than 14580, FIG.
4). Anti-PTX antibodies were present in mice receiving PT-9K/129G
but not PTX FIG. 4).
[0061] In order to determine the effect of boosting, mice were
immunised intranasally twice with either Frg C+PTX, Frg
C+PT-9K/129G, Frg C+CT, or subcutaneously with Frg C adsorbed to
Alhydrogal. 10 .mu.g of Frg C was administered in each case, and 5
.mu.g of PTX PT-9K/129G or CT. Serum samples were obtained 14 days
after the second immunisation. The IgG responses were analysed by
ELISA and the results are shown in FIGS. 5 and 6. In FIGS. 5 and 6
each point represents the mean of 5 mice+1 SEM. As the Figures
show, following boosting, some of the mice that received Frg C
alone I\N seroconverted (2/5). Mice in the Frg C+PTX group also
exhibited an Frg C response following the second dose and this was
greater than that of mice given Frg C alone I\N (FIG. 5) indicating
that PTX had acted as an adjuvant. Also the boosted Frg C+PTX mice
had developed circulating anti-PTX antibodies (FIG. 6). However,
both the anti-Frg C and anti-PTX response were considerably
inferior to that of Frg C+PT-9K/129G mice (FIGS. 5 and 6). The
greatest responses were seen in the Frg C+CT group. The anti-Frg C
response was greater than in S\C immunised mice, anti-Frg C IgG
could still be detected at a serum dilution of 1/800,000. At the
equivalent dilution the anti-CT response had not begun to
titrate.
EXAMPLE 4
Respiratory 1 qA Responses
[0062] The secretory responses were studied in the nasal lavages of
mice immunised twice as described in Example 3. Lavages were taken
14 days after the second immunisation and the IgA responses were
analysed by ELISA. The IgA responses in a 1/5 dilution of nasal
wash are shown in FIGS. 7 and 8 in which each bar represents the
mean of 5 mice+1 SEM. As the Figures show, IgA anti-Frg C was
present in the nasal lavage of all the mice in the Frg C+PTX, Frg
C+PT-9K/129G and Frg C+CT groups (FIG. 7). As in the serum, the
response was greatest in the mice that received CT as adjuvant.
There was very little Frg C specific IgA recovered from the nasal
cavities of the mice given Frg C only I\N or parenterally. In each
group a single mouse exhibited evidence of an IgA response and that
was only detectable in undiluted nasal lavage. The corresponding
IgA responses to PTX, P1-9K/129G and CT were stronger than those
against Frg C and again the anti-CT response was the strongest
(FIG. 8).
EXAMPLE 5
Comparison of the Intranasal Immunogenicity and Adjuvanticity of
PTX and PT-9K/129 in Inbred and Outbred Mice
[0063] To confirm whether the difference observed between PTX and
PT-9K/129G in terms of immunogenicity and adjuvanticity was related
to the source of the PTX, the single dose study in NIH:S mice was
repeated using PTX from a different supplier (NIBSC). Also in order
to examine whether the genetic background of the host influences
the adjuvant and immunogenicity of active and genetically
inactivated PTX the responses in an inbred strain (BALE\c) were
studied as well. BALB\c mice were selected because it has
previously been reported that this strain can mount a serum
response, albeit weak, to parenterally administered pertussis
toxin. Mice were immunised intranasally with a single dose of 10
.mu.g of Frg C alone or combined with PTX or PT-9K/129G, or with
Frg C adsorbed to Alhydrogel, as previously described. Blood serum
samples were taken 16 days later and were analysed by ELISA. In
order to determine whether mice had developed protective immunity,
they were challenged with tetanus toxin 22 days after immunisation
and fatalities recorded for 4 days. The results are shown in FIGS.
9 and 10. In FIGS. 9 and 10 each point represents the mean of 3
mice+SEM.
[0064] In both BALB\c and NIH:S mice the presence of PT-9K/129G
provoked high titre antibodies to fragment C (FIG. 9). In contrast
to the previous study, PTX did have an adjuvant effect on the Frg C
serum response in NIH:S mice and also did so in BALB\c mice. In
both strains of mice, the combination of Frg C and PT-9K/129G
induced a superior serum Frg C response compared to Frg C+PTX,
although the difference was not large. In BALB\c mice, where
comparison was made, the combination of Frg C and PT-9K/129G given
I\N was nearly as effective as S\C immunisation with Frg C adsorbed
to alhydrogel at eliciting a serum response. As previously, NIH:S
mice did not respond to a single 10 .mu.g dose of Frg C I\N. One of
the three BALB\c mice immunised I\N with Frg C alone did mount a
significant serum response, and this accounts for the large error
bars in FIG. 9, and also the protection data (see below).
[0065] Both strains of mice mounted similar serum responses to PTX
and PT-9K/129G. (FIG. 10). The PTX response was measurable but
weak. The PT-9X/129G response was greater by several orders of
magnitude, as was found in earlier study.
[0066] Mice were challenged with tetanus toxin to determine whether
the anti-fragment C antibodies elicited by I\N immunisation were
protective, and the results are shown in Table 2. All of the mice
receiving Frg C+PTX or PT-9K/129G were protected. The single BALB\c
mouse that seroconverted following I\N immunisation with Frg C
alone was protected, the remaining BALB\c mice in this group, the
similarly immunised NIH:S mice and the naive control BALB\c mice
all died.
TABLE-US-00002 TABLE 2 Comparison of seroconversion to fragment C
and protection from tetanus challenge in I\N immunised BALB\c and
NIH:S mice. Frg C + Frg C + Frg C Frg C PTX PT-9K\129G sub\cut
Naive BALB\c Seroconversion 1/3 3/3 3/3 3/3 0/3 (positive\totals)
Protection 1/3 3/3 3/3 3/3 0/3 (survivors/totals) HIH:S Sero
conversion 0/3 3/3 3/3 ND 0/3 (positive\totals) Protection 0/3 3/3
3/3 ND 0/3 (survivors/totals)
[0067] Mice were immunised I\N with a single dose of Frg C, Frg
C+PT, Frg C+PT-9K/129G, Frg C+CT, or Frg C S\C absorbed to
alhydrogel. They received 10 .mu.g of Frg C and 5 .mu.g of the
other proteins. Mice were sample bled 15 days after immunisation
and challenged 22 days after immunisation with 10 LD50 of tetanus
toxin and deaths were recorded for 4 days.
MATERIALS AND METHODS
Aerosol Challenge with B. Pertussis
[0068] Mice were placed in cages on a rotating carousel in a
plastic exposure chamber as described in P. Novotny et al.
Development for Biological Standards, 61, 27 91985. A bacterial
suspension in PBS was prepared from 2- to 3-day old cultures of B.
pertussis BBC26 grown on CW blood agar plates. The mice were
exposed to an aerosol (generated from the bacterial suspension) of
2.times.109 Colony-forming units (CFU) in PBS by a Turret
mouthpiece tubing operated by a System 22 CR60 high-glow compressor
(Medic-Aid) (Pagham, West Sussex, UK) giving a very fine mist at a
dynamic flow of 8.5 litres/min. The generated mist was drawn
through a chamber by a vacuum pump at a passage of ca.12 L of air
per mist mixture per min, which maintained 70% relative humidity in
the chamber. The exposure to aerosol lasted 30 min; a period of 10
min then allowed the chamber to clear.
[0069] The course of the infection was assessed by performing
counts of viable bacteria in lungs. Groups of four mice were
removed at intervals and killed by cervical dislocation, and their
lungs were aseptically removed and homogenised in a Potter-Elvehjem
homogenizer with 2 ml of PBS. Dilutions of the homogenate were
spotted onto Cohen-Wheeler (CW) blood agar plates and the number of
CFU was determined for each set of lungs.
ELISPOT Assay for Specific Antibody Secreting Cells (ASC) in Murine
Lungs
[0070] Local antibody production in the murine lung was determined
using the ELISPOT technique. Lymphocytes were isolated from murine
lungs as follows: Lungs were washed briefly in PBS to remove traces
of blood and then were finely chopped with a scalpel blade 1 ml of
PBS containing 10 mM MgCl.sub.2, 0.5 U/ml collagenase A (Boehringer
Mannheim, Lewes, UK) and 0.25 mg/ml DNase 1 (Boehringer) was added
for each pair of lungs and incubated at 370 C with gentle agitation
for 45 min. The mixture was then passed through a 40 gauge mesh.
Lumps were pressed through the mesh with the plunger from a 5 ml
syringe. The cell suspension was placed in a centrifuge tube and
allowed to stand for several minutes to allow large debris to
settle. The supernatant was removed and the cells were pelleted and
washed several times. Red cells and non-viable cells were removed
by centrifugation on a Ficol-Isopaque gradient (LSM, Flow
Laboratories Ltd, Herts, UK). After washing cell viability was
determined by Trypan Blue exclusion. Cells were finally suspended
in RPM 11640 complete Medium (10% foetal calf serum, penicillin 100
IU/ml, streptomycin 100 g/ml, L-glutamine 2 mm; Flow).
[0071] The ELISPOT assay was performed as follows. Briefly, 24-well
tissue culture plates (Costar) were coated overnight with P.69, FHA
or OVA (0.5 ml of 1 gml in PBA) after washing and blocking 0.5 ml
volumes of dilutions of the lymphocyte suspension in complete RPM1
1640 were added to the wells and incubated at 370 C/10% CO2 for 3
h. After washing goat anti-mouse IgG, A or M (1/1000, Sigma) and
rabbit anti-goat IgG-alkaline phosphatase (1/1000, Sigma) were
added sequentially. Finally, substrate solution (0.51 of 1 mg/ml
5-bromo-4-chloro-3-indolyl phosphate (BCIP) in
2-amino-2-methyl-1,3-propanediol (AMP) buffer, Sigma) was added and
plates were incubated until blue spots were visible under low power
microscopy.
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