U.S. patent application number 08/945750 was filed with the patent office on 2001-07-26 for acellular pertussis vaccines and methods of preparation thereof.
Invention is credited to BARRETO, LUIS, BOUX, LESLIE, FAHIM, RAAFAT E F, HERBERT, ANDREW, JACKSON, GAIL E D, KLEIN, MICHEL H, TAN, LARRY U L, THIPPHAWONG, JOHN, VOSE, JOHN R.
Application Number | 20010009666 08/945750 |
Document ID | / |
Family ID | 27029924 |
Filed Date | 2001-07-26 |
United States Patent
Application |
20010009666 |
Kind Code |
A1 |
VOSE, JOHN R ; et
al. |
July 26, 2001 |
ACELLULAR PERTUSSIS VACCINES AND METHODS OF PREPARATION THEREOF
Abstract
Acellular pertussis vaccines comprise purified toxin or toxoid
thereof, filamentous haemagglutinin, pertactin and fimbrial
agglutinogens formulated to confer protection to at least 70% of
members of an at-risk population. The fimbrial agglutinogens may be
prepared from a Bordetella strain, particularly a B. pertussis
strain, by a multiple step procedure involving extraction of the
fimbrial agglutinogens from cell paste and concentrating and
purifying the extracted material.
Inventors: |
VOSE, JOHN R; (TASSIN LA
DEMI-LUNE, FR) ; FAHIM, RAAFAT E F; (ONTARIO, CA)
; JACKSON, GAIL E D; (ONTARIO, CA) ; TAN, LARRY U
L; (ONTARIO, CA) ; HERBERT, ANDREW; (EAST
YORK, CA) ; BOUX, LESLIE; (QUEBEC, CA) ;
BARRETO, LUIS; (ONTARIO, CA) ; THIPPHAWONG, JOHN;
(MOUNTAIN VIEW, CA) ; KLEIN, MICHEL H; (ONTARIO,
CA) |
Correspondence
Address: |
MICHAEL I STEWART
SIM & MCBURNEY
330 UNIVERSITY AVENUE
6TH FLOOR
ONTARIO
M5G1R7
CA
|
Family ID: |
27029924 |
Appl. No.: |
08/945750 |
Filed: |
June 9, 1998 |
PCT Filed: |
May 2, 1996 |
PCT NO: |
PCT/CA96/00278 |
Current U.S.
Class: |
424/184.1 ;
424/240.1 |
Current CPC
Class: |
A61K 39/00 20130101;
A61K 2039/55505 20130101; A61K 39/099 20130101; A61K 2039/55544
20130101; Y02A 50/466 20180101; A61P 37/00 20180101; C07K 14/235
20130101; A61P 31/04 20180101; A61K 2039/545 20130101 |
Class at
Publication: |
424/184.1 ;
424/240.1 |
International
Class: |
A61K 039/00; A61K
039/38; A61K 039/10 |
Claims
What we claim is:
1. A vaccine composition for protecting an at-risk human population
against a case of disease caused by infection by B. pertussis,
which comprises pertussis toxoid, filamentous haemagglutinin,
pertactin and agglutinogens of B. pertussis in purified form in
selected relative amounts to confer protection to the extent of at
least about 70% of members of the at-risk population.
2. The vaccine of claim 1 wherein said pertussis toxoid is present
in an amount of about 5 to about 30 .mu.g nitrogen, said
filamentous haemagglutinin is present in an amount of about 5 to
about 30 .mu.g nitrogen, said pertactin is present in an amount of
about 3 to about 15 .mu.g nitrogen and said agglutinogens are
present in an amount of about 1 to about 10 .mu.g nitrogen, in a
single human dose.
3. The vaccine of claim 2 containing about 10 .mu.g nitrogen of
pertussis toxoid, about 5 .mu.g nitrogen of filamentous
haemagglutinin, about 5 .mu.g nitrogen of pertactin and about 3
.mu.g nitrogen of agglutinogens in a single human dose.
4. The vaccine of claim 2 containing about 20 .mu.g nitrogen of
pertussis toxoid, about 20 .mu.g nitrogen of filamentous
haemagglutinin, about 5 .mu.g nitrogen of pertactin and about 3
.mu.g nitrogen of agglutinogens in a single human dose.
5. The vaccine of claim 1 wherein the extent of protection is at
least about 80% for a case of pertussis having a spasmodic cough of
duration at least 21 days and confirmed bacterial infection.
6. The vaccine of claim 1 wherein the extent of protection is at
least about 70% for a case of mild pertussis having a cough of at
least one day duration.
7. The vaccine of claim 2 wherein the extent of protection is about
85% for a case having a spasmodic cough of duration at least 21
days and confirmed bacterial infection.
8. The vaccine of claim 1 wherein said agglutinogen comprise
fimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen 3 (Agg 3)
substantially free from agglutinogen 1.
9. The vaccine of claim 8 wherein the weight ratio of Agg 2 to Agg
3 is from about 1.5:1 to about 2:1.
10. The vaccine of claim 1 further comprising tetanus toxoid and
diphtheria toxoid.
11. The vaccine of claim 10 wherein said diphtheria toxoid is
present in an amount of about 15 Lfs and tetanus toxoid is present
In an amount of about 5 Lfs.
12. The vaccine of claim 1 further comprising an adjuvant.
13. The vaccine of claim 12 wherein the adjuvant is alum.
14. A method of immunizing an at-risk human population against
disease caused by infection by B. pertussis, which comprises
administering to members of the at-risk human population an
immunoeffective amount of the vaccine composition of claim 1 to
confer protection to the extent of at least about 70% of the
members of the at-risk population.
15. The use of purified forms of pertussis toxoid, filamentous
haemagglutinin, pertactin and fimbrial agglutinogens of B.
pertussis in the manufacture of a vaccine composition for
administration to an at-risk human population to confer protection
to the extent of at least about 70% of members of said at-risk
human population.
16. The use of claim 15 wherein there is used In the manufacture of
a single human dose of the vaccine composition, from about 5 to
about 30 .mu.g of nitrogen of said pertussis toxoid, about 5 to
about 30 .mu.g of nitrogen.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of copending U.S.
patent application Ser. No. 08/501,743 filed Jul. 12, 1995, which
itself is a continuation-in-part of copending U.S. patent
application Ser. No. 08/433,646 filed May 4, 1995.
FIELD OF INVENTION
[0002] The present invention relates to acellular pertussis
vaccines, components thereof, and their preparation.
BACKGROUND TO THE INVENTION
[0003] Whooping cough or pertussis is a severe, highly contagious
upper respiratory tract infection caused by Bordetella pertussis.
The World Health Organization estimates that there are 60 million
cases of pertussis per year and 0.5 to 1 million associated deaths
(ref. 1. Throughout this specification., various references are
referred to in parenthesis to more fully describe the state of the
art to which this invention pertains. Full bibliographic
information for each citation is found at the end of the
specification, immediately following the claims. The disclosures of
these references are hereby incorporated by reference into the
present disclosure) In unvaccinated populations, a pertussis
incidence rate as high as 80% has been observed in children under 5
years old (ref. 2). Although pertussis is generally considered to
be a childhood disease, there is increasing evidence of clinical
and asymptomatic disease in adolescents and adults (refs. 3, 4 and
5).
[0004] The introduction of whole-cell vaccines composed of
chemically- and heat-inactivated B. pertussis organisms in the
1940's was responsible for a dramatic reduction in the incidence of
whooping cough caused by B. pertussis. The efficacy rates for
whole-cell vaccines have been estimated at up to 95% depending on
case definition (ref. 6) . While infection with B. pertussis
confers life-long immunity, there is increasing evidence for waning
protection after immunization with whole-cell vaccines (ref. 3).
Several reports citing a relationship between whole-cell pertussis
vaccination, reactogenicity and serious side-effects led to a
decline in vaccine acceptance and consequent renewed epidemics
(ref. 7). More recently defined component pertussis vaccines have
been developed.
Antigens for Defined Pertussis Vaccines
[0005] Various acellular pertussis vaccines have been developed and
include the Bordetella pertussis antigens, Pertussis Toxin (PT),
Filamentous haemagglutonin (FHA), the 69 kDa outer membrane protein
(pertactin) and fimbrial agglutinogens (see Table 1 below. The
Tables appear at the end of the specification).
Pertussis Toxin
[0006] Pertussis toxin is an exotoxin which is a member of the A/B
family of bacterial toxins with ADP-ribosyltransferase activity
(ref. 8). The A-moiety of these toxins exhibit the
ADP-ribosyltransferase activity and the B portion mediates binding
of the toxin to host cell receptors and the translocation of A to
its site of action. PT also facilitates the adherence of B.
pertussis to ciliated epithelial cells (ref. 9) and also plays a
role in the invasion of macrophages by B. pertussis (ref. 10).
[0007] All acellular pertussis vaccines have included PT, which has
been proposed as a major virulence factor and protective antigen
(ref. 11, 12). Natural infection with B. pertussis generates both
humoral and cell-mediated responses to PT (refs. 13 to 17). Infants
have transplacentally-derived anti-PT antibodies (refs. 16, 18) and
human colostrum containing anti-PT antibodies was effective in the
passive protection of mice against aerosol infection (ref. 19). A
cell-mediated immune (CMI) response to PT subunits has been
demonstrated after immunization with an acellular vaccine (ref. 20)
and a CMI response to PT was generated after whole-cell vaccination
(ref. 13). Chemically-inactivated PT in whole-cell or component
vaccines is protective in animal models and in humans (ref. 21)
Furthermore, monoclonal antibodies specific for subunit S1 protect
against B. pertussis infection (refs. 22 and 23).
[0008] The main pathophysiological effects of PT are due to its
ADP-ribosyltransferase activity. PT catalyses the transfer of
ADP-ribose from NAD to the G.sub.i guanine nucleotide-binding
protein, thus disrupting the cellular adenylate cyclase regulatory
system (ref. 24). PT also prevents the migration of macrophages and
lymphocytes to sites of inflammation and interferes with the
neutrophil-mediated phagocytosis and killing of bacteria (ref. 25).
A number of in vitro and in vivo assays have been used to asses the
enzymatic activity of S1 and/or PT, including the ADP-ribosylation
of bovine transducin (ref. 26), the Chinese hamster ovary (CHO)
cell clustering assay (ref. 27) , histamine sensitization (ref.
28), leukocytosis, and NAD glycohydrolase. When exposed to PT, CHO
cells develop a characteristic clustered morphology. This
phenomenon is dependent upon the binding of PT, and subsequent
translocation and ADP-ribosyltransferase activity of S1 and thus
the CHO cell clustering assay is widely used to test the integrity
and toxicity of PT holotoxins.
Filamentous Haemagglutonin
[0009] Filamentous haemagglutonin is a large (220 kDa) non-toxic
polypeptide which mediates attachment of B. pertussis to ciliated
cells of the upper respiratory tract during bacterial colonization
(refs. 9, 29). Natural infection induces anti-FHA antibodies and
cell mediated immunity (refs. 13, 15, 17, 30 and 31). Anti-FHA
antibodies are found in human colostrum and are also transmitted
transplacentally (refs. 17, 18 and 19). Vaccination with whole-cell
or acellular pertussis vaccines generates anti-FHA antibodies and
acellular vaccines containing FHA also induce a CMI response to FHA
(refs. 20, 32). FHA is a protective antigen in a mouse respiratory
challenge model after active or passive immunization (refs. 33,
34). However, alone FHA does not protect in the mouse intracerebral
challenge potency assay (ref. 28).
69 kDa Outer Membrane Protein (Pertactin)
[0010] The 69 kDa protein is an outer membrane protein which was
originally identified from B. bronchiseptica (ref. 35). It was
shown to be a protective antigen against B. bronchiseptica and was
subsequently identified in both B. pertussis and B. parapertussis.
The 69 kDa protein binds directly to eukaryotic cells (ref. 36) and
natural infection with B. pertussis induces an anti-P.69 humoral
response (ref. 14) and P.69 also induces a cell-mediated immune
response (ref. 17, 37, 38). Vaccination with whole-cell or
acellular vaccines induces anti-P.69 antibodies (refs. 32, 39) and
acellular vaccines induce P.69 CMI (ref. 39). Pertactin protects
mice against aerosol challenge with B. pertussis (ref. 40) and in
combination with FHA, protects in the intracerebral challenge test
against B. pertussis (ref. 41). Passive transfer of polyclonal or
monoclonal anti-P.69 antibodies also protects mice against aerosol
challenge (ref. 42).
Agglutinogens
[0011] Serotypes of B. pertussis are defined by their agglutinating
fimbriae. The WHO recommends that whole-cell vaccines include types
1, 2 and 3 agglutinogens (Aggs) since they are not cross-protective
(ref. 43). Agg 1 is non-fimbrial and is found on all B. pertussis
strains while the serotype 2 and 3 Aggs are fimbrial. Natural
infection or immunization with whole-cell or acellular vaccines
induces anti-Agg antibodies (refs. 15, 32). A specific
cell-mediated immune response can be generated in mice by Agg 2 and
Agg 3 after aerosol infection (ref. 17). Aggs 2 and 3 are
protective in mice against respiratory challenge and human
colostrum containing anti-agglutinogens will also protect in this
assay (refs. 19, 44, 45).
Acellular Vaccines
[0012] The first acellular vaccine developed was the two-component
PT+FHA vaccine (JNIH 6) of Sato et al. (ref. 46). This vaccine was
prepared by co-purification of PT and FHA antigens from the culture
supernatant of B. pertussis strain Tohama, followed by formalin
toxoiding. Acellular vaccines from various manufacturers and of
various compositions have been used successfully to immunize
Japanese children against whopping cough since 1981 resulting in a
dramatic decrease in incidence of disease (ref. 47). The JNIH 6
vaccine and a mono-component PT toxoid vaccine (JNIH 7) were tested
in a large clinical trial in Sweden in 1986. Initial results
indicated lower efficacy than the reported efficacy of a whole-cell
vaccine, but follow-up studies have shown it to be more effective
against milder disease diagnosed by serological methods (refs. 48,
49, 50, 51). However, there was evidence for reversion to toxicity
of formalin-inactivated PT in these vaccines. These vaccines were
also found to protect against disease rather than infection.
[0013] A number of new acellular pertussis vaccines are currently
being assessed which include combinations of PT, FHA, P.69, and/or
agglutinogens and these are listed in Table 1. Several techniques
of chemical detoxication have been used for PT including
inactivation with formalin (ref. 46), glutaraldehyde (ref. 52),
hydrogen peroxide (ref. 53), and tetranitromethane (ref. 54).
[0014] Thus, current commercially-available acellular pertussis
vaccines may not contain appropriate formulations of appropriate
antigens in appropriate immunogenic forms to achieve a desired
level of efficacy in a pertussis-susceptible human population.
[0015] It would be desirable to provide efficacious accellular
pertussis vaccines containing selected relative amounts of selected
antigens and methods of production thereof.
SUMMARY OF THE INVENTION
[0016] The present invention is directed towards acellular
pertussis vaccine preparations, components thereof, methods of
preparation of such vaccines and their components, and methods of
use thereof.
[0017] In a further aspect of the invention, there is provided an
immunogenic composition comprising the fimbrial agglutinogen
preparation as provided herein. The immunogenic composition may be
formulated as a vaccine for in vivo use for protecting a host
immunized therewith from disease caused by Bordetella and may
comprise at least one other Bordetella antigen. The at least one
other Bordetella antigen may be filamentous haemagglutinin, the 69
kDa outer membrane protein adenylate cyclase, Bordetella
lipooligosaccharide, outer membrane proteins and pertussis toxin or
a toxoid thereof, including genetically detoxified analogs
thereof.
[0018] In a further aspect of the invention, the immunogenic
composition as provided herein may comprise at least one
non-Bordetella immunogen. Such non-Bordetella immunogen may be
diphtheria toxoid, tetanus toxoid, fcapsular pooysaccharide of
Haemophilus, outer membrane protein of Haemoophilus, hepatitis B
surface antigen, polio, mumps, measles and/or rubella.
[0019] The immunogenic compositions as provided herein may further
comprise an adjuvant and such adjuvant may be aluminum phosphate,
aluminum hydroxide, Quil A, QS21, calcium phosphate, calcium
hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester
of an amino acid or a lipoprotein.
[0020] In accordance with one aspect of the present invention,
there is provided a vaccine composition for protecting an at-risk
human population against a case of disease caused by infection by
B. pertussis, which comprises pertussis toxoid, filamentous
haemagglutinin, pertactin and agglutinogens in purified form in
selected relative amounts to confer protection to the extent of at
least about 70% of members of the at-risk population.
[0021] Such vaccine composition may contain about 5 to about 30
.mu.g nitrogen of pertussis toxoid, about 5 to about 30 .mu.g
nitrogen of filamentous haemagglutinin, about 3 to about 15 .mu.g
nitrogen of pertactin and about 1 to about 10 .mu.g nitrogen of
agglutinogens.
[0022] In one specific embodiment, the vaccine may comprise
pertussis toxoid, filamentous haemagglutinin, the 69 kDa protein
and filamentous agglutinogens of Bordetella at a weight ratio of
about 10:5:5:3 as provided by about 10 .mu.g of pertussis toxoid,
about 5 .mu.g of filamentous haemagglutinin, about 5 .mu.g of 69
kDa protein and about 3 .mu.g of fimbrial agglutinogens in a single
human dose. In a further particular embodiment, the vaccine may
comprise pertussis toxoid, filamentous haemagglutinin, 69 kDa
protein and fimbrial agglutinogens in a weight ratio of about
20:20:5:3 as provided by about 20 .mu.g of pertussis toxoid, about
20 .mu.g of filamentous haemagglutinin, about 5 .mu.g of 69 kDa
protein and about 3 .mu.g of fimbrial agglutinogens in a single
human dose. In a yet further particular embodiment, the vaccine may
comprise pertussis toxoid filamentous haemagglutinin, 69 kDa
protein and fimbrial agglutinoaens in a weight ratio of about
20:10:10:6 as provided by about 20 .mu.g of pertussis toxoid, about
10 .mu.g of filamentous haemagglutinin, about 10 .mu.g of 69 kDa
protein and about 6 .mu.g of fimbrial agglutinogens in a single
human dose.
[0023] The extent of protection to the at-risk human population
afforded by the vaccine composition of the invention may be at
least about 80%, preferably about 85%, for a case of spasmodic
cough of duration at least 21 days and culture-confirmed bacterial
infection. The extent of protection to the at-risk human population
may be at least about 70% for a case of mild pertussis having a
cough of at least one day duration.
[0024] The agglutinogens component of the vaccine preferably
comprise fimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen
3 (Agg 3) substantially free from agglutinogen 1. The weight ratio
of Agg 2 to Agg 3 may be from about 1.5:1 to about 2:1.
[0025] The vaccine provided herein may be combined with tetanus
toxoid and diphtheria toxoid to provide a DTP vaccine. In one
embodiment, the vaccine contains about 15 Lfs of diphtheria toxoid
and about 5 Lfs of tetanus toxoid.
[0026] In addition, the vaccine may also comprise an adjuvant,
particularly alum.
[0027] In a further aspect of the present invention, there is
provided a method of immunizing an at-risk human population against
disease caused by infection by B. pertussis, which comprises
administering to members of the at-risk human population an
immunoeffective amount of the vaccine composition provided herein
to confer protection to the extent of at least about 70% of the
members of the at-risk population.
[0028] Advantages of the present invention include an improved
acellular pertussis vaccine composition of increased efficacy.
[0029] The present invention further provides, in an additional
aspect, purified forms of pertussis toxin, filamentous
haemagglutinin, pertactin and fimbrial agglutinogens of B.
pertussis when used in the manufacture of a vaccine composition for
administration to an at-risk human population to confer protection
to the extent of at least about 70% of the members of said at-risk
human population.
[0030] In such use, there may be employed in the manufacture of a
single human dose of the vaccine composition from about 30 .mu.g of
nitrogen of pertactin and about 1 to about 10 .mu.g of nitrogen of
the fimbrial agglutinogens. In particular, the vaccine composition
as provided herein have been selected by the National Institute of
Allergy and Infectious Diseases (NIAID) of the United States
Government for evaluation in a double-blind, human efficacy
clinical trial, thereby establishing a sufficient basis to those
especially skilled in the art that the compositions will be
effective to some degree in preventing the stated disease
(pertussis). The subject of that trial (being a vaccine as provided
herein) has met the burden of being reasonably predictive of
utility.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The present invention will be further understood from the
following detailed description and Examples with reference to the
accompanying drawing in which:
[0032] FIG. 1 is a schematic flow sheet of a procedure for the
isolation of an agglutinogen preparation from a Bordetella
strain.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Referring to FIG. 1, there is illustrated a flow sheet of a
method for preparing an agglutinogen preparation from a Bordetella
strain. As seen in FIG. 1, a Bordetella cell paste containing the
agglutinogens, such as B. pertussis cell paste, is extracted with,
for example, a urea-containing buffer, such as 10 mM potassium
phosphate, 150 mM NaCl and 4M urea, to selectively extract the
agglutinogens from the cell paste to produce a first supernatant
(sp1) containing agglutinogens and a first residual precipitate
(ppt1). The first supernatant (sp1) is separated from the first
residual precipitate (ppt1) such as by centrifugation. The residual
precipitate (ppt1) is discarded. The clarified supernatant (sp1)
then may be concentrated and diafiltered against, for example, 10
mM potassium phosphate/150 mM NaCl/0.1% Triton X-100 using, for
example, a 100 to 300 kDa NMWL membrane filter.
[0034] The first supernatant then is incubated at a temperature and
for a time to produce a clarified supernatant (sp2) containing
agglutinogens and a second discard precipitate (ppt2) containing
non-agglutinogen contaminants. Appropriate temperatures include
about 50.degree. C. to about 100.degree. C., including about
75.degree. to about 85.degree. C., and appropriate incubation times
include about 1 to about 60 minutes. The clarified supernatant then
is concentrated by, for example, the addition of polyethylene
glycol of molecular weight about 8000 (PEG 8000) to a final
concentration of about 4.5.+-.0.2% and stirring gently for a
minimum of about 30 minutes to produce a third precipitate (ppt3)
which may be collected by centrifugation. The remaining supernatant
sp3 is discarded.
[0035] This third precipitate (ppt3) is extracted with, for
example, a buffer comprising 10 mM potassium phosphate/150 mM NaCl
to provide the crude fimbrial agglutinogen-containing solution. 1M
potassium phosphate may be added to the crude fimbrial solution to
make it about 100 mM with respect to potassium phosphate.
Alternatively, the clarified supernatant of heat-treated fimbrial
agglutinogens can be purified without precipitation by
gel-filtration chromatography using a gel, such as Sepharose CL6B.
The fimbrial agglutinogens in the crude solution then are purified
by column chromatography, such as, by passing through a PEI silica
column, to produce the fimbrial agglutinogen preparation in the
run-through.
[0036] This fimbrial agglutinogen containing run-through may be
further concentrated and diafiltered against, for example, a buffer
containing 10 mM potassium phosphate/150 mM NaCl using a 100-300
kDa NMWL membrane. The agglutinogen preparation may be sterilized
by filtration through a .ltoreq.0.22 .mu.M membrane filter, to
provide the final purified fimbrial agglutinogen preparation
containing fimbrial agglutinogen 2 and 3.
[0037] An agglutinogen preparation from a Bordetella strain may
comprise fimbrial agglutinogen 2 (Agg 2) and fimbrial agglutinogen
3 (Agg 3) substantially free from agglutinogen 1. The weight ratio
of Agg 2 to Agg 3 may be from about 1.5:1 to about 2:1. Such
fimbrial agglutinogen preparations may be produced by the method as
provided herein and described in detail above. The present
invention also extends to immunogenic compositions (including
vaccines) comprising the fimbrial agglutinogen preparations
provided as described above. Such vaccines contain other Bordetella
immunogens, including filamentous haemagglutinin, the 69 kDa outer
membrane protein and pertussis toxin or a toxoid thereof, including
genetically detoxified analogs of PT as described in, for example,
ref. 68.
[0038] Such vaccines may include non-Bordetella immunogens
including diphtheria toxoid, tetanus toxoid, capsular
polysaccharide of Haemophilus, outer membrane protein of
Haemophilus, hepatitis B surface antigen, polio, mumps, measles and
rubella.
[0039] Each of the Bordetella antigens is individually absorbed to
adjuvant (such as alum) to provide for convenient and rapid
production of vaccines containing selected relative amounts of
antigens in vaccines as provided herein in order to confer
protection to an extent of at least about 70% of the members of an
at risk population, preferably at least about 80% of such
population.
[0040] In selected embodiments, the invention provides vaccines
with the following characteristics (Ag proteins used herein are
based on Kjedahl test results performed on purified concentrates
and are expressed as /g of protein nitrogen), all of which may be
administered by intramuscular injection:
[0041] (a) CP.sub.10/5/5/3DT
[0042] One formulation of component pertussis vaccine combined with
diphtheria and tetanus toxoids was termed CP.sub.10/5/5/3DT. Each
0.5 ml human dose of CP.sub.10/5/5/3DT was formulated to contain
about:
1 10 .mu.g Pertussis toxoid (PT) 5 .mu.g Filamentous haemagglutonin
(FHA) 5 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB) 3 .mu.g 69 kDa
outer membrane protein 15 Lf Diphtheria toxoid 5 Lf Tetanus toxoid
1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol, as
preservative
[0043] (b) CP.sub.20/20/5/3DT
[0044] Another formulation of component pertussis vaccine combined
with diphtheria and tetanus toxoids was termed CP.sub.20/20/5/3DT.
Each 0.5 ml human dose of CP.sub.20/20/5/3DT was formulated to
contain about:
2 20 .mu.g Pertussis toxoid (PT) 20 .mu.g Filamentous
haemagglutonin (FHA) 5 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB)
3 .mu.g 69 kDa outer membrane protein 15 Lf Diphtheria toxoid 5 Lf
Tetanus toxoid 1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol, as
preservative
[0045] (c) CP.sub.10//5/5DT
[0046] One formulation of component pertussis vaccine combined with
diphtheria and tetanus toxoids was termed CP.sub.10/5/5DT. Each 0.5
mL human dose of CP.sub.10/5/5 was formulated to contain about:
3 10 .mu.g Pertussis toxoid (PT) 5 .mu.g Filamentous haemagglutonin
(FHA) 5 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB) 15 Lf
Diphtheria toxoid 5 Lf Tetanus toxoid 1.5 mg Aluminum phosphate
0.6% 2-phenoxyethanol as preservative
[0047] (d) CP.sub.20/10/10/6DT
[0048] A further formulation of component pertussis vaccine
combined with diphtheria and tetanus toxoids was termed
CP.sub.20/10/10/6DT. Each 0.5 ml human dose of CP.sub.20/10/10/6DT
was formulated to contain about:
4 20 .mu.g Pertussis toxoid (PT) 10 .mu.g Filamentous
haemagglutonin (FHA) 10 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB)
6 .mu.g 69 kDa outer membrane protein (69 kDA) 15 Lf Diphtheria
toxoid 5 Lf Tetanus toxoid 1.5 mg Aluminum phosphate 0.6%
2-phenoxyethanol, as preservative
[0049] The other Bordetella immunogens, pertussis toxin (including
genetically detoxified analogs thereof, as described in, for
example, Klein et al, U.S. Pat. No. 5,085,862 assigned to the
assignee hereof and incorporated herein by reference thereto), FHA
and the 69 kDa protein may be produced by a variety of methods such
as described below:
Purification of PT
[0050] PT may be isolated from the culture supernatant of a B.
pertussis strain using conventional methods. For example, the
method of Sekura et al (ref. 55) may be used. PT is isolated by
first absorbing culture supernatant onto a column containing the
dye-ligand gel matrix, Affi-Gel Blue (Bio-Rad Laboratories,
Richmond, Calif.). PT is eluted from this column by high salt, such
as, 0.75 M magnesium chloride and, after removing the salt, is
passed through a column of fetuin-Sepharose affinity matrix
composed of fetuin linked to cyanogen bromide-activated Sepharose.
PT is eluted from the fetuin column using 4M magnesium salt.
[0051] Alternatively, the method of Irons et al (ref. 56) may be
used. Culture supernatant is absorbed onto a CNBr-activated
Sepharose 4B column to which haptoglobin is first covalently bound.
The PT binds to the absorbent at pH 6.5 and is eluted from the
column using 0.1M Tris/0.5M NaCl buffer by a stepwise change to pH
10.
[0052] Alternatively, the method described in U.S. Pat. No.
4,705,686 granted to Scott et al on Nov. 10, 1987 and incorporated
herein by reference thereto may be used. In this method culture
supernatants or cellular extracts of B. pertussis are passed
through a column of an anion exchange resin of sufficient capacity
to adsorb endotoxin but permit Bordetella antigens to flow through
or otherwise be separated from the endotoxin.
[0053] Alternatively, PT may be purified by using perlite
chromatography, as described in EP Patent No. 336 736, assigned to
the assignee thereof and incorporated herein by reference
thereto.
Detoxification of PT
[0054] PT is detoxified to remove undesired activities which could
cause side reactions of the final vaccine. Any of a variety of
conventional chemical detoxification methods can be used, such as
treatment with formaldehyde, hydrogen peroxide, tezranitro-methane,
or glutaraldehyde.
[0055] For example, PT can be detoxified with glutaraldehyde using
a modification of the procedure described in Munoz et al (ref. 57).
In this detoxification process purified PT is incubated in a
solution containing 0.01 M phosphate buffered saline. The solution
is made 0.05% with glutaraldehyde and the mixture is incubated at
room temperature for two hours, and then made 0.02 M with L-lysine.
The mixture is further incubated for two hours at room temperature
and then dialyzed for two days against 0.01 M PBS. In a particular
embodiment, the detoxification process of EP Patent No. 336 736 may
be used. Briefly PT may be detoxified with glutaraldehyde as
follows:
[0056] Purified PT in 75 mM potassium phosphate at pH 8.0
containing 0.22M sodium chloride is diluted with an equal volume of
glycerol to protein concentrations of approximately 50 to 400
.mu.g/ml. The solution is heated to 37.degree. C. and detoxified by
the addition of glutaraldehyde to a final concentration of 0.5%
(w/v). The mixture is kept at 37.degree. C. for 4 hrs and then
aspartic acid (1.5 M) is added to a final concentration of 0.25 M.
The mixture is incubated at room temperature for 1 hour and then
diafiltered with 10 volumes of 10 mM potassium phosphate at pH 8.0
containing 0.15M sodium chloride and 5% glycerol to reduce the
glycerol and to remove the glutaraldehyde. The PT toxoid is
sterile-filtered through a 0.2 .mu.M membrane.
[0057] If recombinant techniques are used to prepare a PT mutant
molecule which shows no or little toxicity, for use as the toxoided
molecule, chemical detoxification is not necessary.
Purification of FHA
[0058] FHA may be purified from the culture supernatant essentially
as described by Cowell et al (ref. 58). Growth promoters, such as
methylated beta-cyclodextrins, may be used to increase the yield of
FHA in culture supernatants. The culture supernatant is applied to
a hydroxylapatite column. FHA is adsorbed onto the column, but PT
is not. The column is extensively washed with Triton X-100 to
remove endotoxin. FHA is then eluted using 0.5M NaCl in 0.1M sodium
phosphate and, if needed, passed through a fetuin-Sepharose column
to remove residual PT. Additional purification can involve passage
though a Sepharose CL-6B column.
[0059] Alternatively, FHA may be purified using monoclonal
antibodies to the antigen, where the antibodies are affixed to a
CNBr-activated affinity column (ref. 59).
[0060] Alternatively, FHA may be purified by using perlite
chromatography as described in the above-mentioned EP 336 736.
Purification of 69 kDa Outer Membrane Protein (pertactin)
[0061] The 69 kDa outer membrane protein (69K or pertactin) may be
recovered from bacterial cells by first inactivating the cells with
a bacteriostatic agent, such as thimerosal, as described in
published EP 484 621 and incorporated herein by reference thereto.
The inactivated cells are suspended in an aqueous medium, such as
PBS (pH 7 to 8) and subjected to repeated extraction at elevated
temperature (45 to 60.degree. C.) with subsequent cooling to room
temperature or 4.degree. C. The extractions release the 69K protein
from the cells. The material containing the 69K protein is
collected by precipitation and passed through an Affi-gel Blue
column. The 69K protein is eluted with a high concentration of
salt, such as G.5M magnesium chloride. After dialysis, it is passed
through a chromatofocusing support.
[0062] Alternatively, the 69 kDa protein may be purified from the
culture supernatant of a B. pertussis culture, as described in
published POT Application WO 91/15505, in the name of the assignee
hereof and incorporated herein by reference thereto.
[0063] Other appropriate methods of purification of the 69 kDa
outer membrane protein from B. pertussis are described in U.S. Pat.
No. 5,276,142, granted to Gotto et al on Jan. 4, 1984 and in U.S.
Pat. No. 5,101,014, granted to Burns on Mar. 31, 1992.
[0064] A number of clinical trials were performed in humans as
described herein to establish the safety, non-reactogenicity and
utility of component vaccines for protection against pertussis. In
particular, immune responses to each of the antigens contained in
the vaccines (as shown, for example, in Table 3 below) were
obtained. One particular acellular pertussis vaccine
CP.sub.10/5/5/3DT was analyzed in a large placebo-controlled,
multi-centre, double-randomized clinical trial in an at-risk human
population to estimate the efficacy of the vaccine against typical
pertussis.
[0065] The case definition for typical pertussis disease was:
[0066] Twenty-one days or more of spasmodic cough, and either
[0067] culture-confirmed B. pertussis, or
[0068] serological evidence of Bordetella specific infection
indicated by a 100% IgG or IgA antibody rise in ELISA against FHA
or PT in paired sera, or
[0069] if serological data is lacking, the study child has been in
contact with a case of culture-confirmed B. pertussis in the
household with onset of cough within 28 days before or after the
onset of cough in the study child.
[0070] The results of this study showed CP.sub.10/5/5/3DT to be
about 85% efficacious in preventing pertussis as defined in the
case definition for typical pertussis disease as described above.
In the same study, a two-component pertussis acellular vaccine
containing only PT and FHA was about 58% efficacious and a
whole-cell pertussis vaccine was about 48% efficacious (see Table 4
below). In addition, the CP.sub.10/5/5/3DT vaccine prevented mild
pertussis defined as a cough of at least one day duration to an
efficacy of about 77%. In particular, the profile of immune
response obtained was substantially the same as that obtained
following immunization with whole-cell pertussis vaccines which are
reported to be highly efficacious against pertussis.
Vaccine Preparation and Use
[0071] Thus, immunogenic compositions, suitable to be used as
vaccines, may be prepared from the Rordetella immunogens as
disclosed herein. The vaccine elicits an immune response in a
subject which produces antibodies that may be opsonizing or
bactericidal. Should the vaccinated subject be challenged by B.
pertussis, such antibodies bind to and inactivate the bacteria.
Furthermore, opsonizing or bactericidal antibodies may also provide
protection by alternative mechanisms.
[0072] Immunogenic compositions including vaccines may be prepared
as injectibles, as liquid solutions or emulsions. The Bordetella
immunogens may be mixed with pharmaceutically acceptable excipients
which are compatible with the immunogens. Such excipients may
include water, saline, dextrose, glycerol, ethanol, and
combinations thereof. The immunogenic compositions and vaccines may
further contain auxiliary substances, such as wetting or
emulsifying agents, pH buffering agents, or adjuvants to enhance
the effectiveness thereof. Immunogenic compositions and vaccines
may be administered parenterally, by injection subcutaneously or
intramuscularly. The immunogenic preparations and vaccines are
administered in a manner compatible with the dosage formulation,
and in such amount as will be therapeutically effective,
immunogenic and protective. The quantity to be administered depends
on the subject to be treated, including, for example, the capacity
of the immune system of the individual to synthesize antibodies,
and, if needed, to produce a cell-mediated immune response. Precise
amounts of active ingredient required to be administered depend on
the judgment of the practitioner. However, suitable dosage ranges
are readily determinable by one skilled in the art and may be of
the order of micrograms of the immunogens. Suitable regimes for
initial administration and booster doses are also variable, but may
include an initial administration followed by subsequent
administrations. The dosage may also depend on the route of
administration and will vary according to the size of the host.
[0073] The concentration of the immunogens in an immunogenic
composition according to the invention is in general about 1 to
about 95%. A vaccine which contains antigenic material of only one
pathogen is a monovalent vaccine. Vaccines which contain antigenic
material of several pathogens are combined vaccines and also belong
to the present invention. Such combined vaccines contain, for
example, material from various pathogens or from various strains of
the same pathogen, or from combinations of various pathogens.
[0074] Immunogenicity can be significantly improved if the antigens
are co-administered with adjuvants, commonly used as 0.005 to 0.5
percent solution in phosphate buffered saline. Adjuvants enhance
the immunogenicity of an antigen but are not necessarily
immunogenic themselves. Adjuvants may act by retaining the antigen
locally near the site of administration to produce a depot effect
facilitating a slow, sustained release of antigen to cells of the
immune system. Adjuvants can also attract cells of the immune
system to an antigen depot and stimulate such cells to elicit
immune responses.
[0075] Immunostimulatory agents or adjuvants have been used for
many years to improve the host immune responses to, for example,
vaccines. Intrinsic adjuvants, such as lipopolysaccharides,
normally are the components of the killed or attenuated bacteria
used as vaccines. Extrinsic adjuvants are immunomodulators which
are typically non-covalently linked to antigens and are formulated
to enhance the host immune responses. Thus, adjuvants have been
identified that enhance the immune response to antigens delivered
parenterally. Some of these adjuvants are toxic, however, and can
cause undesirable side-effects, making them unsuitable for use in
humans and many animals. Indeed, only aluminum hydroxide and
aluminum phosphate (collectively commonly referred to as alum) are
routinely used as adjuvants in human and veterinary vaccines. The
efficacy of alum in increasing antibody responses to diphtheria and
tetanus toxoids is well established and, more recently, a HBsAg
vaccine has been adjuvanted with alum. While the usefulness of alum
is well established for some applications, it has limitations. For
example, alum is ineffective for influenza vaccination and
inconsistently elicits a cell mediated immune response. The
antibodies elicited by alum-adjuvanted antigens are mainly of the
IgGl isotype in the mouse, which may not be optimal for protection
by some vaccinal agents.
[0076] A wide range of extrinsic adjuvants can provoke potent
immune responses to antigens. These include saponins complexed to
membrane protein antigens (immune stimulating complexes), pluronic
polymers with mineral oil, killed mycobacteria in mineral oil,
Freund's complete adjuvant, bacterial products, such as muramyl
dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A,
and liposomes.
[0077] To efficiently induce humoral immune responses (HIR) and
cell-mediated immunity (CMI), immunogens are often emulsified in
adjuvants. Many adjuvants are toxic, inducing granulomas, acute and
chronic inflammations (Freund's complete adjuvant, FCA), cytolysis
(saponins and Pluronic polymers) and pyrogenicity, arthritis and
anterior uveitis (LPS and MDP) . Although FCA is an excellent
adjuvant and widely used in research, it is not licensed for use in
human or veterinary vaccines because of its toxicity.
[0078] Desirable characteristics of ideal adjuvants include:
[0079] (1) lack of toxicity;
[0080] (2) ability to stimulate a long-lasting immune response;
[0081] (3) simplicity of manufacture and stability in long-term
storage;
[0082] (4) ability to elicit both CMI and HIR to antigens
administered by various routes;
[0083] (5) synergy with other adjuvants;
[0084] (6) capability of selectively interacting with populations
of antigen presenting cells (APC):
[0085] (7) ability to specifically elicit appropriate T.sub.H.sup.1
or T.sub.H.sup.2 cell-specific immune responses; and
[0086] (8) ability to selectively increase appropriate antibody
isotype levels (for example, IgA) against antigens.
[0087] U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8,
1989 which is incorporated herein by reference thereto teaches
glycolipid analogues including N-glycosylamides, N-glycosylureas
and N-glycosylcarbamates, each of which is substituted in the sugar
residue by an amino acid, as immuno-modulators or adjuvants. Thus,
Lockhoff et al. (U.S. Pat. No. 4,855,283 and ref. 60) reported that
N-glycolipid analogs displaying structural similarities to the
naturally-occurring glycolipids, such as glycosphingolipids and
glycoglycerolipids, are capable of eliciting strong immune
responses in both herpes simplex virus vaccine and pseudorabies
virus vaccine. Some glycolipids have been synthesized from long
chain alkylamines and fatty acids that are linked directly with the
sugars through the anomeric carbon atom, to mimic the functions of
the naturally occurring lipid residues.
[0088] U.S. Pat. No. 4,258,029 granted to Moloney, assigned to the
assignee hereof and incorporated herein by reference thereto,
teaches that octadecyl tyrosine hydrochloride (OTH) functions as an
adjuvant when complexed with tetanus toxoid and formalin
inactivated type I, II and III poliomyelitis virus vaccine. Also,
Nixon-George et al. (ref. 61), reported that octodecyl esters of
aromatic amino acids complexed with a recombinant hepatitis B
surface antigen, enhanced the host immune responses against
hepatitis B virus.
EXAMPLES
[0089] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific Examples. These Examples are
described solely for the purposes of illustration and are not
intended to limit the scope of the invention. Changes in form and
substitution of equivalents are contemplated as circumstances may
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitation.
[0090] Methods of protein biochemistry, fermentation and immunology
used but not explicitly described in this disclosure and these
Examples are amply reported in the scientific literature and are
well within the ability of those skilled in the art.
Example 1
[0091] This Example describes the growth of Bordetella
pertussis.
Master Seed
[0092] Master seed cultures of a Bordetella pertussis strain were
held as freeze-dried seed lots, at 2.degree. C. to 8.degree. C.
Working Seed
[0093] The freeze-dried culture was recovered in Hornibrook medium
and used to seed Bordet-Gengou Agar (BGA) plates. Hornibrook medium
has the following composition:
5 Component for 1 liter Casein hydrolysate (charcoal treated) 10.0
g Nicotinic acid 0.001 g Calcium chloride 0.002 g Sodium chloride
5.0 g Magnesium chloride hexahydrate 0.025 g Potassium chloride
0.200 g Potassium phosphate dibasic 0.250 g Starch 1.0 g Distilled
water to 1.0 liter
[0094] The pH is adjusted to 6.9.+-.0.1 with lo sodium carbonate
solution. The medium is dispensed into tubes and sterilized by
steaming in the autoclave for 20 minutes and autoclaving for 20
minutes at 121.degree. C. to 124.degree. C. The seed was
subcultured twice, firstly on BGA plates then on Component
Pertussis Agar (CPA). Component Pertussis Agar (CPA) has the
following composition:
6 NaCl 2.5 g/L KH.sub.2PO.sub.4 0.5 g/L KCl 0.2 g/L
MgCl.sub.2(H.sub.2O).sub.6 0.1 g/L Tris base 1.5 g/L Casamino acids
10.0 g/L NaHGlutamate 10.0 g/L Conc. HCl to pH 7.2 Agar 15.0 g/L
Growth factors (CPGF) 10.0 mL/L
[0095] Component Pertussis Growth Factors (CPGF)--100.times. have
the following composition:
7 L-cysteine HCl 4.0 g/L Niacin 0.4 g/L Ascorbic acid 40.0 g/L
Glutathione, reduced 15.0 g/L Fe.sub.2SO.sub.4, (H.sub.2O).sub.7
1.0 g/L Dimethyl-.beta.-cyclodextrin 100 g/L
CaCl.sub.2(H.sub.2O).sub.2 2.0 g/L
[0096] The final culture was suspended in Pertussis Seed Suspension
Buffer (CPSB), dispensed into 2 to 4 ml aliquots and stored frozen
at -60.degree. C. to -85.degree. C. Pertussis Seed Suspension
Buffer (PSSB) has the following composition:
8 Casamino acids 10.0 g/L Tris base 1.5 g/L Anhydrous glycerol 100
mL/L Conc. HCl to pH 7.2
[0097] These glycerol suspensions provided the starting material
for the preparation of the working seed.
Cultivation Process
[0098] Propagation of the working seed was conducted in Component
Pertussis Agar Roux bottles for 4 to 7 days at 34.degree. C. to
38.degree. C. Following this cultivation, cells were washed off
agar with Component Pertussis Broth (CPB). Samples were observed by
Gram stain, for culture purity and opacity.
[0099] Cells were transferred to 4 liter conical flasks containing
CPB and incubated at 34.degree. C. to 38.degree. C. for 20 to 26
hours with shaking. Samples were observed by Gram stain and culture
purity was checked. Flasks were pooled and the suspension was used
to seed two fermenters containing CPB (10 liter volume starting at
OD.sub.600 0.1-0.4). The seed was grown to a final OD.sub.600 of
5.0 to 10.0. Samples were tested by Gram strain, for culture
purity, by antigen specific ELISAs and for sterility.
Example 2
[0100] This Example describes the purification of antigens from the
Bordetella pertussis cell culture.
Production of Broth and Cell Concentrates
[0101] Bacterial suspension was grown in two production fermenters,
at 34.degree. C. to 37.degree..degree.C. for 35 to 50 hours. The
fermenters were sampled for media sterility testing. The suspension
was fed to a continuous-flow disk-stack centrifuge (12,000.times.g)
to separate cells from the broth. Cells were collected to await
extraction of fimbriae component. The clarified liquor was passed
through .ltoreq. 0.22 .mu.m membrane filter. The filtered liquor
was concentrated by ultra filtration using a 10 to 30 kDa nominal
molecular weight limit (NMWL) membrane. The concentrate was stored
to await separation and purification of the Pertussis Toxin (PT),
Filamentous haemagglutonin (FHA) and 69 kDa (pertactin)
components.
Separation of the Broth Components
[0102] The broth components (69 kDa, PT and FHA) were separated and
purified by perlite chromatography and selective elution steps,
essentially as described in EP Patent No. 336 736 and applicants
published PCT Application No. WO 91/15505, described above. The
specific purification operations effected are described below.
Pertussis Toxin (PT)
[0103] The perlite column was washed with 50 mM Tris, 50 mM
Tris/0.5% Triton X-100 and 50 mM Tris buffers. The PT fraction was
eluted from the perlite column with 50 mM Tris/0.12M NaCl
buffer.
[0104] The PT fraction from the perlite chromatography was loaded
onto a hydroxyLapatite column and then washed with 30 mM potassium
phosphate buffer. PT was eluted with 75 mM potassium phosphate/225
mM NaCl buffer. The column was washed with 200 mM potassium
phosphate/0.6M NaCl to obtain the FHA fraction which was discarded.
Glycerol was added to the purified PT to 50% and the mixture was
stored at 2.degree. C. to 8.degree. C. until detoxification, within
one week.
Filamentous Haemagglutonin (FRA)
[0105] The FHA fraction was eluted from the perlite column with 50
mM Tris/0.6M NaCl. Filamentous haemagglutinin was purified by
chromatography over hydroxylapatite. The FHA fraction from the
perlite column was loaded onto a hydroxylapatite column then washed
with 30 mM potassium phosphate containing 0.56 Triton X-100,
followed by 30 mM potassium phosphate buffer. The PT fraction was
eluted with 85 mM potassium phosphate buffer and discarded. The FHA
fraction was then eluted with 200 mM potassium phosphate/0.6M NaCl
and stored at 2.degree. C. to 8.degree. C. until detoxification
within one week.
69 kDa (pertactin)
[0106] The broth concentrate was diluted with water for injection
(WFI) to achieve a conductivity of 3 to 4 mS/cm and loaded onto a
perlite column at a loading of 0.5 to 3.5 mg protein per ml
perlite. The run-through (69 kDa Component Fraction) was
concentrated by ultrafiltration using a 10 to 30 kDa NMWL membrane.
Ammonium sulphate was added to the run-through concentrate to 35%
.+-.3% (w/v) and the resulting mixture stored at 2.degree. C. to
8.degree. C. for 4.+-.2 days or centrifuged (7,000.times.g)
immediately. Excess supernatant was decanted and the precipitate
collected by centrifugation (7,000.times.g). The 69 kDa pellet was
either stored frozen at -20.degree. C. to -30.degree. C. or
dissolved in Tris or phosphate buffer and used immediately.
[0107] The 69 kDa outer membrane protein obtained by the 35% (w/v)
ammonium sulphate precipitation of concentrated perlite run-through
was used for the purification. Ammonium sulphate (100.+-.5 g per
liter) was added to the 69 kDa fraction and the mixture stirred for
at least 2 hours at 2.degree. C. to 8.degree. C. The mixture was
centrifuged (7,000.times.g) to recover the supernatant. Ammonium
sulphate (100 to 150 g per liter) was added to the supernatant and
the mixture stirred for at least 2 hours at 2.degree. C. to
8.degree. C. The mixture was centrifuged (7,000.times.g) to recover
the pellet, which was dissolved in 10 mM Tris, HCl, pH 8. The ionic
strength of the solution was adjusted to the equivalent of 10 mM
Tris HCl (pH 8), containing 15 mM ammonium sulphate.
[0108] The 69 kDa protein was applied to a hydroxylapatite column
connected in tandem with a Q-Sepharose column. The 69 kDa protein
was collected in the run-through, was flushed from the columns with
10 mM Tris, HCl (pH 8), containing 15 mM ammonium sulphate and
pooled with 69 kDa protein in the run-through. The 69 kDa protein
pool was diafiltered with 6 to 10 volumes of 10 mM potassium
phosphate (pH 8), containing 0.15M NaCl on a 100 to 300 kDa NMWL
membrane. The ultra filtrate was collected and the 69 kDa protein
in the ultra filtrate concentrated.
[0109] The 69 kDa protein was solvent exchanged into 10 mM Tris HCl
(pHB), and adsorbed onto Q-Sepharose, washed with 10 mM Tris HCl
(pH 8)/5 mM ammonium sulphate. The 69 kDa protein was eluted with
50 mM potassium phosphate (pH 8). The 69 kDa protein was
diafiltered with 6 to 10 volumes of 10 mM potassium phosphate (pH
8) containing 0.15M NaCl on a 10 to 30 kDa NMWL membrane. The 69
kDa protein was sterile filtered through a .ltoreq.0.22 .mu.m
filter. This sterile bulk was stored at 2.degree. C. to 8.degree.
C. and adsorption was performed within three months.
Fimbrial Agglutinogens
[0110] The agglutinogens were purified from the cell paste
following separation from the broth. The cell paste was diluted to
a 0.05 volume fraction of cells in a buffer containing 10 mM
potassium phosphate, 150 mM NaCl and 4M urea and was mixed for 30
minutes. The cell lysate was clarified by centrifugation
(12,000.times.g) then concentrated and diafiltered against 10 mM
potassium phosphate/150 mM NaCl/0.1% Triton X-100 using a 100 to
300 kDa NMWL membrane filter.
[0111] The concentrate was heat treated at 80.degree. C. for 30 min
then reclarified by centrifugation (9,000.times.g). PEG 8000 was
added to the clarified supernatant to a final concentration of 4.5%
.+-.0.2% and stirred gently for a minimum of 30 minutes. The
resulting precipitate was collected by centrifugation
(17,000.times.g) and the pellet extracted with 10 mM potassium
phosphate/150 mM NaCl buffer to provide a crude fimbrial
agglutinogen solution. The fimbrial agglutinogens were purified by
passage over PEI silica. The crude solution was made 100 mM with
respect to potassium phosphate using 1M potassium phosphate buffer
and passed through the PEI silica column.
[0112] The run-through from the columns was concentrated and
diafiltered against 10 mM potassium phosphate/150 mM NaCl buffer
using a 100 to 300 kDa NMWL membrane filter. This sterile bulk is
stored at 2.degree. C. to 8.degree. C. and adsorption performed
within three months. The fimbrial agglutinogen preparation
contained fimbrial Agg 2 and fimbrial Agg 3 in a weight ratio of
about 1.5 to about 2:1 and was found to be substantially free from
Agg 1.
Example 3
[0113] This Example describes the toxoiding of the purified
Bordetella pertussis antigens, PT and FHA.
[0114] PT, prepared in pure form as described in Example 2, was
toxoided by adjusting the glutaraldehyde concentration in the PT
solution to 0.5% .+-.0.1% and incubating at 37.degree.
C..+-.3.degree. C. for 4 hours. The reaction was stopped by adding
L-aspartate to 0.21.+-.0.02M. The mixture was then held at room
temperature for 1.+-.0.1 hours and then at 2.degree. C. to
8.degree. C. for 1 to 7 days.
[0115] The resulting mixture was diafiltered against 10 mM
potassium phosphate/0.15M NaCl/5% glycerol buffer on a 30 kDa NMWL
membrane filter and then sterilized by passage through a
.ltoreq.0.22 .mu.m membrane filter. This sterile bulk was stored at
2.degree. C. to 8.degree. C. and adsorption performed within three
months.
[0116] The FHA fraction, prepared in pure form as described in
Example 2, was toxoided by adjusting the L-lysine and formaldehyde
concentration to 47.+-.5 mM and 0.24.+-.0.05% respectively and
incubating at 35.degree. C. to 38.degree. C. for 6 weeks. The
mixture was then diafiltered against 10 mM potassium phosphate/0.5M
NaCl using a 30 kDa NMWL membrane filter and sterilized by passage
through a membrane filter. This sterile bulk was stored a 2.degree.
C. to 8.degree. C. and adsorption performed within three
months.
Example 4
[0117] This Example describes the adsorption of the purified
Bordetella pertussis antigens.
[0118] For the individual adsorption of PT, FHA, Agg and 69 kDa
onto aluminum phosphate (alum), a stock solution of aluminum
phosphate was prepared to a concentration of 18.75.+-.1 mg/ml. A
suitable vessel was prepared and any one of the antigens
aseptically dispensed into the vessel. 2-phenoxyethanol was
aseptically added to yield a final concentration of 0.6% .+-.0.1%
v/v and stirred until homogeneous. The appropriate volume of
aluminum phosphate was aseptically added into the vessel. An
appropriate volume of sterile distilled water was added to bring
the final concentration to 3 mg aluminum phosphate/ml. Containers
were sealed and labelled and allowed to stir at room temperature
for 4 days. The vessel was then stored awaiting final
formulation.
Example 5
[0119] This Example describes the formulation of a component
pertussis vaccine combined with diphtheria and tetanus toxoids.
[0120] The B. pertussis antigens prepared as described in the
preceding Examples were formulated with diphtheria and tetanus
toxoids to provide several component pertussis (CP) vaccines.
[0121] The pertussis components were produced from Bordetella
pertussis grown in submerged culture as described in detail in
Examples 1 to 4 above. After completion of growth, the culture
broth and the bacterial cells were separated by centrifugation.
Each antigen was purified individually. Pertussis toxin (PT) and
Filamentous Haemagglutinin (FHA) were purified from the broth by
sequential chromatography over perlite and hydroxylapatite. PT was
detoxified with glutaraldehyde and any residual PT (approximately
1%) present in the FHA fraction was detoxified with formaldehyde.
Fimbrial Agglutinogens (2+3) (AGG) were prepared from the bacterial
cells. The cells were disrupted with urea and heat treated, and the
fimbrial agglutinogens were purified by precipitation with
polyethylene glycol and chromatography over polyethyleneimine
silica. The 69 kDa protein (pertactin) component was isolated from
the run through from the perlite chromatography step (Example 2) by
ammonium sulphate precipitation, and purified by sequential
chromatography over hydroxylapatite and Q-sepharose. All components
were sterilized by filtration through a 0.22 .mu.m membrane
filter.
[0122] Diphtheria toxoid was prepared from Corynebacterium
diphtheriae grown in submerged culture by standard methods. The
production of Diphtheria Toxoid is divided into five stages, namely
maintenance of the working seed, growth of Corynebacterium
diphtheriae, harvest of Diphtheria Toxin, detoxification of
Diphtheria Toxin and concentration of Diphtheria Toxoid.
Preparation of Diphtheria Toxoid
[0123] (I) Working Seed
[0124] The strain of Corynebacterium diphtheriae was maintained as
a freeze-dried seed lot. The reconstituted seed was grown on
Loeffler slopes for 18 to 24 hours at 35.degree. C..+-.2.degree.
C., and then transferred to flasks of diphtheria medium. The
culture was then tested for purity and Lf content. The remaining
seed was used to inoculate a fermenter.
[0125] (II) Growth of Corynebacterium diphtheriae
[0126] The culture was incubated at 35.degree. C..+-.2.degree. C.
and agitated in the fermenter. Predetermined amounts of ferrous
sulphate, calcium chloride and phosphate solutions were added to
the culture. The actual amounts of each solution (phosphate,
ferrous sulphate, calcium chloride) were determined experimentally
for each lot of medium. The levels chosen are those which gave the
highest Lf content. At the end of the growth cycle (30 to 50
hours), the cultures were sampled for purity, and Lf content.
[0127] The pH was adjusted with sodium bicarbonate, and the culture
inactivated with 0.4% toluene for 1 hour at a maintained
temperature of 35.degree. C..+-.2.degree. C. A sterility test was
then performed to confirm the absence of live C. diphtheriae.
[0128] (III) Harvest of Diphtheria Toxin
[0129] The toluene treated cultures from one or several fermenters
were pooled into a large tank. Approximately 0.12% sodium
bicarbonate, 0.25% charcoal, and 23% ammonium sulphate were added,
and the pH was tested.
[0130] The mixture was stirred for about 30 minutes. Diatomaceous
earth was added and the mixture pumped into a depth filter. The
filtrate was recirculated until clear, then collected, and sampled
for Lf content testing. Additional ammonium sulphate was added to
the filtrate to give a concentration of 40%. Diatomaceous earth was
also added. This mixture was held for 3 to 4 days at 2.degree. C.
to 8.degree. C. to allow the precipitate to settle. Precipitated
toxin was collected and dissolved in 0.9% saline. The diatomaceous
earth was removed by filtration and the toxin dialysed against 0.9%
saline, to remove the ammonium sulphate. Dialysed toxin was pooled
and sampled for Lf content and purity testing.
[0131] (IV) Detoxification of Diphtheria Toxin
[0132] Detoxification takes place immediately following dialysis.
For detoxification, the toxin was diluted so that the final
solution contained:
[0133] a) diphtheria toxin at 1000.+-.10% Lf/ml.
[0134] b) 0.5% sodium bicarbonate
[0135] c) 0.5% formalin
[0136] d) 0.9% w/v L-lysine monohydrochloride
[0137] The solution was brought up to volume with saline and the pH
adjusted to 7.6.+-.0.1.
[0138] Toxoid was filtered through cellulose diatomaceous earth
filter pads and/or a membrane prefilter and 0.2 .mu.m membrane
filter into the collection vessel and incubated for 5 to 7 weeks at
34.degree. C. A sample was withdrawn for toxicity testing.
[0139] (V) Concentration of Purified Toxoid
[0140] The toxoids were pooled, then concentrated by
ultrafiltration, and collected into a suitable container. Samples
were taken for Lf content and purity testing. The preservative
(2-phenoxyethanol) was added to give a final concentration of
0.375% and the pH adjusted to 6.6 to 7.6.
[0141] The toxoid was sterilized by filtration through a prefilter
and a 0.2 .mu.m membrane filter (or equivalent) and collected. The
sterile toxoid was then sampled for irreversibility of toxoid Lf
content, preservative content, purity (nitrogen content), sterility
and toxicity testing. The sterile concentrated toxoid was stored at
2.degree. C. to 8.degree. C. until final formulation.
Preparation of Tetanus Toxoid
[0142] Tetanus toxoid (T) was prepared from Clostridium tetani
grown in submerged culture.
[0143] The production of Tetanus Toxoid can be divided into five
stages, namely maintenance of the working seed, growth of
Clostridium tetani, harvest of Tetanus Toxin, detoxification of
Tetanus Toxin and purification of Tetanus Toxoid.
[0144] (I) Working Seed
[0145] The strain of Clostridium tetani used in the production of
tetanus toxin for the conversion to tetanus toxoid was maintained
in the lyophilized form in a seed-lot. The seed was inoculated into
thioglycollate medium and allowed to grow for approximately 24
hours at 35.degree. C..+-.2.degree. C. A sample was taken for
culture purity testing.
[0146] (II) Growth of Clostridium tetani
[0147] The tetanus medium was dispensed into a fermenter,
heat-treated and cooled. The fermenter was then seeded and the
culture allowed to grow for 4 to 9 days at 34.degree. C..+-.
2.degree. C. A sample was taken for culture purity, and Lf content
testing.
[0148] (III) Harvest of Tetanus Toxin
[0149] The toxin was separated by filtration through cellulose
diatomaceous earth pads, and the clarified toxin then
filter-sterilized using membrane filters. Samples were taken for Lf
content and sterility testing. The toxin was concentrated by
ultrafiltration, using a pore size of 30,000 daltons.
[0150] (IV) Detoxification of Tetanus Toxin
[0151] The toxin was sampled for Lf content testing prior to
detoxification. The concentrated toxin (475 to 525 Lf/ml) was
detoxified by the addition of 0.5% w/v sodium bicarbonate, 0.3% v/v
formalin and 0.9% w/v L-lysine monohydrochloride and brought up to
volume with saline. The pH was adjusted to 7.5.+-.0.1 and the
mixture incubated at 37.degree. C. for 20 to 30 days. Samples were
taken for sterility and toxicity testing.
[0152] (V) Purification of Toxoid
[0153] The concentrated toxoid was sterilized through pre-filters,
followed by 0.2 .mu.m membrane filters. Samples were taken for
sterility and Lf content testing.
[0154] The optimum concentration of ammonium sulphate was based on
a fractionation "S" curve determined from samples of the toxoid.
The first concentration was added to the toxoid (diluted to
1900-2100 Lf/ml). The mixture was kept for at least 1 hour at
20.degree. C. to 25.degree. C. and the supernatant collected and
the precipitate containing the high molecular weight fraction,
discarded.
[0155] A second concentration of ammonium sulphate was added to the
supernatant for the second fractionation to remove the low
molecular weight impurities. The mixture was kept for at least 2
hours at 20.degree. C. to 25.degree. C. and then could be held at
2.degree. C. to 8.degree. C. for a maximum of three days. The
precipitate, which represents the purified toxoid, was collected by
centrifugation and filtration.
[0156] Ammonium sulphate was removed from the purified toxoid by
diafiltration, using Amicon (or equivalent) ultrafiltration
membranes with PBS until no more ammonium sulphate could be
detected in the toxoid solution. The pH was adjusted to 6.6. to
7.6, and 2-phenoxyethanol added to give a final concentration of
0.375%. The toxoid was sterilized by membrane filtration, and
samples are taken for testing (irreversibility of toxoid, Lf
content, pH, preservative content, purity, sterility and
toxicity).
[0157] One formulation of a component pertussis vaccine combined
with diphtheria and tetanus toxoids was termed CP.sub.10/5/5/3DT,
Each 0.5 ml human dose of CP.sub.10/5/5/3DT was formulated to
contain:
9 10 .mu.g Pertussis toxoid (PT) 5 .mu.g Filamentous haemagglutonin
(FHA) 5 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB) 3 .mu.g 69 kDa
outer membrane protein 15 Lf Diphtheria toxoid 5 Lf Tetanus toxoid
1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol as preservative
[0158] Another formulation of component pertussis vaccine combined
with diphtheria and tetanus toxoids was termed CP.sub.10/5/5DT.
Each 0.5 ml human dose of CP.sub.10/5/5DT was formulated to
contain:
10 10 .mu.g Pertussis toxoid (PT) 5 .mu.g Filamentous
haemagglutonin (FHA) 5 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB)
15 Lf Diphtheria toxoid 5 Lf Tetanus toxoid 1.5 mg Aluminum
phosphate 0.6% 2-phenoxyethanol as preservative
[0159] Another formulation of Component Pertussis vaccine combined
with diphtheria and tetanus toxoids was termed CP.sub.20/20/5/3DT.
Each 0.5 ml human dose of CP.sub.20/20/5/3DT was formulated to
contain:
11 20 .mu.g Pertussis toxiod (PT) 20 .mu.g Filamentous
haemagglutonin (FHA) 5 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB)
3 .mu.g 69 kDa outer membrane protein 15 Lf Diphtheria toxoid 5 Lf
Tetanus toxoid 1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol as
preservative
[0160] A further formulation of a component pertussis vaccine
combined with diphtheria and tetanus toxoids was termed
CP.sub.20/10/10/6DT. Each 0.5 ml human dose of CP.sub.20/10/10/6DT
was formulated to contain:
12 20 .mu.g Pertussis toxiod (PT) 10 .mu.g Filamentous
haemagglutonin (FHA) 10 .mu.g Fimbrial agglutinogens 2 and 3 (FIMB)
6 .mu.g 69 kDa outer membrane protein 15 Lf Diphtheria toxoid 5 Lf
Tetanus toxoid 1.5 mg Aluminum phosphate 0.6% 2-phenoxyethanol as
preservative
Example 6
[0161] This Example describes the clinical assessment of Component
Acellular Pertussis vaccines, produced in accordance with the
invention.
[0162] (a) Studies in Adults
[0163] Studies in adults and children aged 16 to 20 months
indicated the multi-component vaccines containing fimbrial
agglutinogens to be safe and immunogenic (Table 2).
[0164] A Phase I clinical study was performed in 17 and 18 month
old children in Calgary, Alberta with the five Component Pertussis
vaccine (CP.sub.10/5/5/3DT) and the adverse reaction reported.
Thirty-three children received the vaccine and additionally 35
received the same vaccine without the 69 kDa protein component.
[0165] Local reactions were rare. Systemic adverse reactions,
primarily consisting of irritability were present in approximately
half of study participants, regardless of which vaccine was given.
Significant antibody rises were measured for anti-PT, anti-FHA,
anti-fimbrial agglutinogens and anti-69 kDa IgG antibodies by
enzyme immunoassay and anti-PT antibodies in the CHO cell
neutralization test. No differences in antibody response were
detected in children who received the four component
(CP.sub.10/5/5DT) or five component (CP.sub.10/5/5/3DT) except in
the anti-69 kDa antibody. Children who received the five component
vaccine containing the 69 kDa protein had a significantly higher
post-immunization anti-69 kDa antibody level.
[0166] A dose-response study was undertaken with the 4 component
vaccine in Winnipeg, Manitoba, Canada. Two component vaccine
formulations were used: CP.sub.10/5/5/3DT and CP.sub.20/10/10/6DT,
A whole-cell DPT vaccine was also included as a control.
[0167] This study was a double-blind study in 91, 17 to 18 month
old infants at the time of their booster pertussis dose. Both
CP.sub.10/5/5/3DT and CP.sub.20/10/10/6DT were well tolerated by
these children. No differences were demonstrated in the number of
children who had any local reaction, or systemic reactions after
either of the component vaccines. In contrast, significantly more
children who received the whole-cell vaccine had local and systemic
reactions than those who received the CP.sub.20/10/10/6DT component
vaccines.
Studies in Infants
[0168] Phase II:
[0169] A study was conducted using the CP.sub.10/5/5/3DT vaccine in
Calgary, Alberta and British Columbia, Canada. In this study, 432
infants received the component pertussis vaccine or the whole-cell
control vaccine DPT at 2, 4 and 6 months of age. The
CP.sub.10/5/5/3DT vaccine was well tolerated by these infants.
Local reactions were less common with the component vaccine than
the whole cell vaccine after each dose.
[0170] A significant antibody response to all antigens was
demonstrated after vaccination with the component pertussis
vaccine. Recipients of the whole-cell vaccine had a vigorous
antibody response to fimbrial agglutinogens, D and T. At seven
months, 82% to 89% of component vaccine recipients and 92% of whole
cell vaccine recipients had a four-fold increase or greater rise in
antibody titer to fimbrial agglutinogens. In contrast, antibody
response to FHA was 756 to 78% in component vaccines compared to
31% of whole-cell recipients. A four-fold increase in anti-69 kDa
antibody was seen in 90% to 93% of component vaccines and 75% of
whole-cell recipients. A four-fold rise in antibody against PT by
enzyme immunoassay was seen in 40% to 49% of component vaccines and
32% of whole-cell vaccines; a four-fold rise in PT antibody by CHO
neutralization was found in 55% to 69% of component and 6% of
whole-cell vaccines. (Table 2).
[0171] Phase IIB:
[0172] The CP.sub.20/20/5/3DT and CP.sub.10/10/5/3DT vaccines were
assessed in a randomized blinded study against a D.sub.15PT control
with a lower diphtheria content of 15 Lf compared to a 25 Lf
formulation of 100 infants at 2, 4 and 6 months of age. No
differences in rates of adverse reactions were detected between the
two components formulations; both were significantly less
reactogenic than the whole-cell control. Higher antibody titers
against PT by enzyme immunoassay and CHO neutralization and FHA
were achieved in recipients of the CP.sub.20/20/5/3DT vaccine with
increased antigen content. At 7 months, the anti-FHA geometric mean
titer was 95.0 in CP.sub.20/20/5/3DT recipients, 45.2 in
CP.sub.10/5/5/3DT recipients were only 8.9 in D.sub.15PT
recipients. Anti-PT titers were 133.3, 58.4 and 10.4 by immunoassay
and 82.4, 32.7 and 4.0 by CHO neutralization respectively (Table
2).
[0173] This study demonstrated that the Component Pertussis vaccine
combined with diphtheria and tetanus toxoids adsorbed, with
increased antigen content, was safe and immunogenic in infants and
that the increased antigen content augmented the immune response to
the prepared antigens (PT and FHA) without an increase in
reactogenicity.
[0174] NIAID, PHASE II. U.S. Comparative Trial:
[0175] A phase II study was performed in the United States under
the auspices of the National Institute of Allergy and Infectious
Diseases (NIAID) as a prelude to a large scale efficacy trial of
acellular pertussis vaccines. One component pertussis vaccine of
the invention in combination with diphtheria and tetanus toxoids
adsorbed (CP.sub.10/5/5/3DT) was included in that trial along with
12 other acellular vaccines and 2 whole-cell vaccines. Safety
results were reported on 137 children immunized at 2, 4 and 6
months of age with the CP.sub.10/5/5/3DT component vaccine.
[0176] As seen in previous studies, the component vaccine was found
to be safe, of low reactogenicity and to be well tolerated by
vaccines.
[0177] At 7 months, anti-PT antibody, anti-FHA antibody, anti-69
kDa antibody and anti-fimbrial agglutinogens antibody were all
higher than or equivalent to levels achieved after the whole-cell
vaccines (ref 71 and Table 2). A double blind study was performed
in which children were randomly allocated to receive either the
CP.sub.20/20/5/3DT or the CP.sub.10/5/5/3DT vaccine formulation. A
total of 2050 infants were enrolled in the United States and
Canada; 1961 infants completed the study. Both vaccine formulations
were safe, of low reactogenicity and immunogenic in these infants.
Immunogenicity was assessed in a subgroup of 292. An antibody rise
was elicited to all antigens contained in the vaccine by both
vaccine formulations. The CP.sub.20/20/5/3DT formulation induced
higher antibody titers against FHA but not PT. The
CP.sub.10/5/5/3DT formulation elicited higher titers against
fimbriae and higher agglutinogen titers.
[0178] A further safety and immunogenicity study was conducted in
France. The study design was similar to the North American study,
described above, except that vaccines were administered at 2, 3 and
4 months of age. Local and systemic reactions were generally minor.
Overall the vaccine was well accepted by the French study
participants using this administration regime.
[0179] Placebo-controlled efficacy trial of two acellular pertussis
vaccines and of a whole-cell vaccine in 10,000 infants
[0180] Following the results of the NIAID Phase II U.S. comparative
trial, a two-component and a five-component acellular vaccine were
selected for a multi-centre, controlled, double-randomized
placebo-controlled efficacy trial. The clinical trial was performed
in Sweden, where there is a high incidence of pertussis. The
two-component vaccine contained glyceraldehyde and formalin
inactivated PT (25 kg), formalin treated FHA (25 .mu.g) and
diphtheria toxoid 17 Lf and tetanus toxoid 10 Lf. The
five-component pertussis vaccine was CP.sub.10/5/5/3DT. For the
trial, ten thousand infants, representing approximately one-half
the infants of this age group in Sweden, were recruited in 14
geographically defined study sites by use of birth registry.
[0181] Children born in January and February 1992 were randomized
into a 3-armed trial. After parental consent, two-thirds of the
infants received one out of the two diphtheria-tetanus-acellular
pertussis preparations at two, four and six months of agen. The
control group received DT only. In May 1992, a U.S. Licensed
commercially-available whole-cell DTP vaccine was introduced and
children born in March through December 1992 were randmized into a
4-armed trial. After parental consent, three-quarters of the
infants received one out of three DTP preparations at two, four and
six months of age. The control group received DT only.
[0182] Each vaccine was administered to about 2,500 children.
Vaccines were administered in three doses. The first dose was given
at 2 months of age and not later than 3 months of age. Subsequent
doses were given with 8 week intervals. Vaccines were given by
intramuscular injection.
[0183] The children and their households were followed for 30
months. If pertussis was suspected, clinical data was collected,
and laboratory verification sought by nasal aspirates for
bacteriological culture and polymerase chain reaction (PCR)
diagnosis. Acute and convalescent blood samples were collected for
serological diagnosis.
[0184] Prior to this study, the extent of pertactin afforded by
component pertussis vaccines of the present invention in an at-risk
human population (particularly neonates) was unknown. In
particular, the contribution of the various Bordetella components
and their presence in pertussis vaccines in selected relative
amounts to efficacy of the vaccines was not known.
[0185] The main aim of the trial was to estimate the ability of
acellular pertussis vaccines and whole-cell vaccine to protect
against typical pertussis as compared to placebo.
[0186] A secondary end-point was to explore vaccine efficacy
against confirmed pertussis infection of varying severity.
[0187] Vaccine efficacy is defined as the per cent reduction in the
probability of contracting pertussis among vaccine recipients
relative to unvaccinated children.
[0188] The relative risk of pertussis in two vaccine groups is
expressed as the ratio of the disease probability in the two
groups.
[0189] The probability of contracting pertussis, also called the
attack rate, can be estimated in different ways. In the
calculations of the sample size, the probability of contracting
pertussis in a given study group is estimated by the quotient
between the number of children with pertussis and the children
remaining in the study group at the termination of study
follow-up.
[0190] The efficacy of the component vaccine CP.sub.10/5/5/3DT in
this trial in preventing typical pertussis is shown in Table 4 and
was about 85%. In the same trial, a two-component pertussis
acellular vaccine containing only PT and FHA was about 58%
efficacious and a whole-cell vaccine was about 48% efficacious. The
CP.sub.10/5/5/3DT was also effective in preventing mild pertussis
at an estimated efficacy of about 77%.
SUMMARY OF DISCLOSURE
[0191] In summary of this disclosure, the present invention
provides novel preparations of fimbrial agglutinogens of Bordetella
pertussis and methods for their production. The fimbrial
agglutinogens can be formulated with other Bordetella and
non-Bordetella antigens to produce a number of multi-component
pertussis vaccines. Such vaccines are safe, non-reactogenic,
immunogenic and protective in humans. Modifications are possible
within the scope of this invention.
13TABLE 1 Acellular Pertussis Vaccines Toxoiding Refer- Vaccine PT
Agent FHA P.69 AGG2 AGG3 ence AMVC + H.sub.2O.sub.2.sup.a - - - -
62 Mass PHL.sup.b + TMN.sup.c - - - - 63 Institut + GI.sup.d + - -
- 64 Mrieux Smith-Kline + FI.sup.e/GI + - - - 32 + FI/GI + + - - 32
CAMR.sup.f + FI + - + + 65 Lederle/ + FI + + + - 66 Takeda
Connaught + GI + - + + 32 + GI + + + + 67 .sup.aHydrogen peroxide
inactivated. .sup.bMassachusetts Public Health Laboratories.
.sup.cTNM, tetranitromethane-inactiva- ted. .sup.dGI,
glutaraldehyde-inactivated. .sup.eFI, formalin-inactivated.
.sup.fCentre for Applied Microbiology and Research.
[0192]
14TABLE 2 IgG antibody responses to pertussis antigen and
diphtheria and tetanus toxoids in adults and young children after
Immunization with placebo or acellular pertussis (AP),
diphtheria-tetanus-pertussis (DTP), or multicomponent acellular DTP
(ADTP) toxoids. Adults Children Before immunization
Postimmunization day 28 Before immunization After immunization AP
AP ADTP ADTP Placebo CP.sub.10/5/5/3 Placebo CP.sub.10/5/3 DTP
CP.sub.10/10/5/3DT DTP CP.sub.10/10/5/3DT Pertussis 16.45 22.78
16.56 415.87 43.71 15.45 221.32 306.55 toxoid (9.46-28.62)
(12.11-42.86) (9.08-30.22) (243.91- (14.29-133.88) (8.50-28.10)
(99.83-490.67) (155.84- 709.09) 603.03) Filamentous 15.24 23.59
13.36 317.37 2.93 3.86 30.06 29.86 hemagglutinin (10.28-22.60)
(15.59-35.69) (7.71-23.16) (243.05- (1.81-4.73) (3.03-4.93)
(11.82-76.46) (16.51-53.99) 141.41) Agglutinogens 21.26 28.64 27.0
2048.00 26.72 29.24 315.2 1243.3 (12.14-37.23) (12.20-67.21)
(15.37-47.78) (1025.62- (16.94-42.15) (13.63-62.75) (127.4-779.9)
(594.8-2603.5) 4089.55) Pertactin 7.89 11.47 7.46 855.13 6.54 9.45
60.13 116.16 (4.00-15.56) (6.41-20.55) (3.51-15.87) (396.41-
(2.79-15.33) (5.50-16.23) (24.59-147.04) (57.87-233.19) 1844.67)
CHO cell 12.30 21.11 10.78 604.67 27.47 9.71 270.60 342.51
neutralizing (6.97-21.68) (10.35-43.06) (5.54-20.97) (403.82-
(7.36-102.62) (4.71-20.03) (24.6-1100.8) (146.6-800.2) assay
405.41) Diphtheria <0.1 <0.1 <0.1 <0.1 <0.1 <0.1
8.75 9.65 toxoid (6.52-23.92) (5.62-16.57) Tetanus <0.1 <0.1
<0.1 <0.1 <0.1 <0.1 4.11 6.32 toxoid (3.20-5.28)
(5.31-7.53) No. studied 16 15 16 15 10 25 12 25 Data are expressed
as geometric mean with 95% confidence intervals. For pertussis
toxoid, filamentous hemagglutinin, agglutinogens, perlactin, and
diphtheria and tetanus toxoids, antibody titers expressed as ELISA
units/nL. For CHO cell neutralizing assay, values reflect
reciprocal of highest dilution demonstrating 80%
neutralization.
[0193]
15TABLE 3 Serologic Results or Acellular Pertussis Vaccines In
Infants (2, 4 and 6 Months Old) Geometric Mean Titres CHO Cell
Clinical Number of Fimbrial Neutraliz- Trial Product Study
Participants PT FHA 69 kDa agglutinogens ation Agglutination Tet
Dip 1 CP.sub.10/5/5DT U.S. NIAID 108 38 37 3 229 160 85 7.8 0.8
CP.sub.10/5/5/3DT Multicentre 113 36 36 113 241 150 73 5.0 0.4
Whole Cell (Mass.) Comparative Study 95 20 51 101 70 80 42 - -
Whole Cell (Lederle) (Cycle I) 312 67 3 64 193 270 84 - - 2
CP.sub.10/5/5/3DT Phase II 315 87.1 50.2 29.9 239.8 29.6 - 1.5 0.3
Whole Cell (CLL) Canada 101 20 4.7 6.4 603.2 2.6 - 1.2 0.4 3
CP.sub.10/5/5/1DT Phase IIB 32 58.4 45.2 40.6 111.4 32.7 - 1.0 0.14
CP.sub.20/20/5/1DT Canada 33 133.3 95.0 37.1 203.8 82.4 1.1 0.21
Whole Cell (CLL) 30 10.4 8.9 6.8 393.9 4.0 1.8 0.31 4
CP.sub.10/5/5/1DT Phase IIC 42 105.1 82.5 71.1 358.6 66.9 307.0 2.0
0.33 CP.sub.20/20/5/3DT Canada 250 101.6 163.9 87.6 220.6 68.7
219.2 1.8 0.38 5 CP.sub.20/20/5/3DT Montreal 58 212.7 83.4 106.3
601.9 109.6 - 1.9 0.53 Whole Cell (CLL) Feasibility Study 58 101.4
11.7 16.8 906.9 6.0 1.1 0.27 6 CP.sub.10/5/5DT U.S. NIAID 80 42 34
50 310 196 185 CP.sub.20/20/5/3DT Comparative Study 80 39 87 43 184
254 137 - - Whole Cell (CLI) (Cycle II) 80 2 3 9 33 54 167 Whole
Cell (Lederle) 80 18 2 16 129 137 86 CLI - Connaught Laboratories
Incorporated, Swiftwater, Pennsylvania. CLL - Connaught
Laboratories Limited, Willowdale, Ontario. Mass - Massachusetts
Public Laboratories. Lederle - Lederle Laboratories Inc.
[0194]
16TABLE 4 Efficacy of Acellular Pertussis Vaccines Efficacy %
Vaccine A B CP.sub.10/5/5/3DT 84.7 (80.3.fwdarw.88.5).sup.1 77
PT.sub.25.FHA.sub.25DT 58 (49.8.fwdarw.64.8).sup.1 DPT.sup.2 47.9
(37.1.fwdarw.56.9).sup.1 A: case definition: 21 day spasmodic cough
and culture positive B: case definition: mild pertussis cough of at
least one day Note .sup.1confidence limits .sup.2whole cell
pertussis vaccine
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