U.S. patent application number 15/975388 was filed with the patent office on 2018-09-13 for combination vaccines with lower doses of antigen and/or adjuvant.
This patent application is currently assigned to GlaxoSmithKline Biologicals SA. The applicant listed for this patent is GlaxoSmithKline Biologicals SA. Invention is credited to Barbara BAUDNER, Derek O'HAGAN, Manmohan SINGH, David A.G. SKIBINSKI.
Application Number | 20180256695 15/975388 |
Document ID | / |
Family ID | 45852630 |
Filed Date | 2018-09-13 |
United States Patent
Application |
20180256695 |
Kind Code |
A1 |
BAUDNER; Barbara ; et
al. |
September 13, 2018 |
COMBINATION VACCINES WITH LOWER DOSES OF ANTIGEN AND/OR
ADJUVANT
Abstract
Combination vaccine compositions as well as methods for their
manufacture have a relatively low amount of antigen and/or a
relatively low amount of aluminium, but they can nevertheless have
immunogenicity which is comparable to combination vaccines with a
relatively high amount of antigen and/or a relatively high amount
of aluminium. Aluminium-free combination vaccine compositions are
also provided e.g. compositions which are adjuvanted with an
oil-in-water emulsion adjuvant.
Inventors: |
BAUDNER; Barbara; (Siena,
IT) ; SKIBINSKI; David A.G.; (Seattle, WA) ;
SINGH; Manmohan; (Boston, MA) ; O'HAGAN; Derek;
(Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GlaxoSmithKline Biologicals SA |
Rixensart |
|
BE |
|
|
Assignee: |
GlaxoSmithKline Biologicals
SA
Rixensart
BE
|
Family ID: |
45852630 |
Appl. No.: |
15/975388 |
Filed: |
May 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15887203 |
Feb 2, 2018 |
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15975388 |
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14002700 |
Jan 10, 2014 |
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PCT/IB2012/050989 |
Mar 2, 2012 |
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15887203 |
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61448226 |
Mar 2, 2011 |
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61565980 |
Dec 1, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55505
20130101; A61K 2039/545 20130101; A61K 39/39 20130101; Y02A 50/30
20180101; A61K 39/12 20130101; A61K 2039/55566 20130101; A61K
31/4375 20130101; A61K 31/66 20130101; A61K 39/102 20130101; A61P
31/04 20180101; C12N 2730/10134 20130101; A61K 39/116 20130101;
A61K 39/295 20130101; Y02A 50/466 20180101; C12N 2770/32634
20130101; A61K 39/0018 20130101; A61K 2039/55555 20130101; A61K
2039/70 20130101; A61P 31/12 20180101; A61K 33/06 20130101; A61K
33/06 20130101; A61K 2300/00 20130101; A61K 39/116 20130101; A61K
2300/00 20130101; A61K 39/295 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/39 20060101 A61K039/39; A61K 31/66 20060101
A61K031/66; A61K 33/06 20060101 A61K033/06; A61K 39/102 20060101
A61K039/102; A61K 39/116 20060101 A61K039/116; A61K 39/12 20060101
A61K039/12; A61K 31/4375 20060101 A61K031/4375; A61K 39/295
20060101 A61K039/295 |
Claims
1. An immunogenic composition comprising (i) an oil-in-water
emulsion adjuvant (ii) a diphtheria toxoid, a tetanus toxoid, a
pertussis toxoid, and a Hib conjugate (iii) a hepatitis B virus
surface antigen and/or an inactivated poliovirus antigen.
2. The composition of claim 1, wherein the composition is
aluminium-free.
3. The composition of claim 1, wherein the oil-in-water emulsion
adjuvant has oil droplets with a sub-micron diameter.
4. The composition of claim 1, wherein the oil-in-water emulsion
adjuvant includes squalene and/or polysorbate 80.
5. The composition of claim 1, further comprising conjugated
capsular saccharide from one or more of meningococcal serogroups A,
C, W135 and/or Y.
6. The composition of claim 1, further comprising conjugated
capsular saccharide from one or more of pneumococcal serotypes 1,
2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,
18C, 19A, 19F, 20, 22F, 23F and/or 33F.
7. The composition of claim 1, further comprising (i) a
meningococcal factor H binding protein antigen and/or (ii) a
Neisserial Heparin Binding Antigen and/or (iii) a meningococcal
NhhA antigen and/or (iv) a meningococcal outer membrane vesicle.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Division of co-pending U.S.
application Ser. No. 15/877,203, filed Feb. 2, 2018, which is a
continuation of U.S. application Ser. No. 14/002,700, filed on Jan.
10, 2014, which is the U.S. National Phase of International
Application No. PCT/IB2012/050989, filed on Mar. 2, 2012 and
published in English; which claims priority to U.S. Provisional
Application No. 61/448,226, filed on Mar. 2, 2011 and U.S.
Provisional Application No. 61/565,980, filed on Dec. 1, 2011. The
teachings of the above applications are incorporated herein in
their entirety by reference.
TECHNICAL FIELD
[0002] This invention is in the field of combination vaccines i.e.
vaccines containing mixed immunogens from more than one pathogen,
such that administration of the vaccine can simultaneously immunize
a subject against more than one pathogen.
BACKGROUND ART
[0003] Vaccines containing antigens from more than one pathogenic
organism within a single dose are known as "multivalent" or
"combination" vaccines. Various combination vaccines have been
approved for human use in the EU and the USA, including trivalent
vaccines for protecting against diphtheria, tetanus and pertussis
("DTP" vaccines) and trivalent vaccines for protecting against
measles, mumps and rubella ("MMR" vaccines). Combination vaccines
offer patients the advantage of receiving a reduced number of
injections, which can lead to the clinical advantage of increased
compliance (e.g. see chapter 29 of reference 1), particularly for
pediatric vaccination.
[0004] Current combination vaccines can include relatively high
amounts of aluminium salts as adjuvants which causes concern to
some patient pressure groups despite empirical safety studies
[2,3]. For instance, the aluminium levels in known combination
vaccines are as follows (see also Table A below):
TABLE-US-00001 Trade name Antigens Al.sup.+++ content per unit dose
Pediacel D-T-Pa-Hib-IPV 0.33 mg Pediarix D-T-Pa-HBV-IPV
.ltoreq.0.85 mg Pentacel D-T-Pa-Hib-IPV 0.33 mg Tritanrix-HepB
D-T-Pw-HBV 0.63 mg Quinvaxem D-T-Pw-Hib-HBV 0.3 mg Hexavac
D-T-Pa-IPV-Hib-HBV 0.3 mg Boostrix (USA) D-T-Pa .ltoreq.0.39 mg
[0005] A vaccine with lower levels of aluminium would be helpful
for some patient groups, and it is an object of the present
invention to provide such vaccines, ideally without loss of vaccine
potency.
[0006] Another drawback with current vaccines is that they require
relatively high amounts of antigen, whereas various documents show
that protective effects might be achieved with lower amounts of
antigen e.g. reference 4 shows that the amount of Hib antigen can
be halved in a D-T-Pw-Hib vaccine without loss of immunological
response, and reference 5 argues that a reduced IPV dose can be
used while maintaining an adequate level of protection against
polio. It is an object of the present invention to provide further
vaccines with reduced amounts of antigen, ideally without loss of
immunoprotective effect.
SUMMARY OF THE INVENTION
[0007] The invention provides a variety of combination vaccine
compositions as well as methods for their manufacture. Typically
the compositions have a relatively low amount of antigen and/or a
relatively low amount of aluminium, but they can nevertheless have
immunogenicity which is comparable to combination vaccines with a
relatively high amount of antigen and/or a relatively high amount
of aluminium. Aluminium-free combination vaccine compositions are
also provided e.g. compositions which are adjuvanted with an
oil-in-water emulsion adjuvant.
[0008] In a first embodiment the invention provides an immunogenic
composition in a unit dose form for administration to a patient
comprising (i) a diphtheria toxoid, a tetanus toxoid, and a
pertussis toxoid, and (ii) an aluminium salt adjuvant, wherein the
amount of Al.sup.+++ in the unit dose is less than 0.2 mg.
[0009] The invention also provides an immunogenic composition
comprising (i) a diphtheria toxoid, a tetanus toxoid, and a
pertussis toxoid and (ii) an aluminium salt adjuvant, wherein the
concentration of Al.sup.+++ is less than 0.4 mg/ml.
[0010] In a second embodiment the invention provides an immunogenic
composition comprising (i) an aluminium salt adjuvant and (ii) a
low dose of each of a diphtheria toxoid, a tetanus toxoid, and a
pertussis toxoid.
[0011] In a third embodiment the invention provides an immunogenic
composition in a unit dose form for administration to a patient
comprising (i) a low dose of each of a diphtheria toxoid, a tetanus
toxoid, and a pertussis toxoid, and (ii) an aluminium salt
adjuvant, wherein the amount of Al.sup.+++ in the unit dose is less
than 0.2 mg.
[0012] The invention also provides an immunogenic composition
comprising (i) a low dose of each of a diphtheria toxoid, a tetanus
toxoid, and a pertussis toxoid and (ii) an aluminium salt adjuvant,
wherein the concentration of Al.sup.+++ is less than 0.4 mg/ml.
[0013] In a fourth embodiment the invention provides an immunogenic
composition comprising (i) an oil-in-water emulsion adjuvant (ii) a
diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, and a Hib
conjugate (iii) a hepatitis B virus surface antigen and/or an
inactivated poliovirus antigen. The composition is ideally
aluminium-free.
[0014] The aluminium salt adjuvant advantageously has an adsorbed
TLR agonist, as discussed below.
[0015] A further aspect of the invention is an immunisation
schedule for an infant in which only one or two DTaP-containing
compositions are administered. This aspect is explained in further
detail below.
[0016] Diphtheria Toxoid
[0017] Diphtheria is caused by Corynebacterium diphtheriae, a
Gram-positive non-sporing aerobic bacterium. This organism
expresses a prophage-encoded ADP-ribosylating exotoxin (`diphtheria
toxin`), which can be treated (e.g. using formaldehyde) to give a
toxoid that is no longer toxic but that remains antigenic and is
able to stimulate the production of specific anti-toxin antibodies
after injection. Diphtheria toxoids are disclosed in more detail in
chapter 13 of reference 1. Preferred diphtheria toxoids are those
prepared by formaldehyde treatment. The diphtheria toxoid can be
obtained by growing C. diphtheriae in growth medium (e.g. Fenton
medium, or Linggoud & Fenton medium), which may be supplemented
with bovine extract, followed by formaldehyde treatment,
ultrafiltration and precipitation. The toxoided material may then
be treated by a process comprising sterile filtration and/or
dialysis.
[0018] Quantities of diphtheria toxoid can be expressed in
international units (IU). For example, the NIBSC [6] supplies the
`Diphtheria Toxoid Adsorbed Third International Standard 1999`
[7,8], which contains 160 IU per ampoule. As an alternative to the
IU system, the `Lf` unit ("flocculating units", the "limes
flocculating dose", or the "limit of flocculation") is defined as
the amount of toxoid which, when mixed with one International Unit
of antitoxin, produces an optimally flocculating mixture [9]. For
example, the NIBSC supplies `Diphtheria Toxoid, Plain` [10], which
contains 300 Lf per ampoule and `The 1st International Reference
Reagent For Diphtheria Toxoid For Flocculation Test` [11] which
contains 900 Lf per ampoule. The concentration of diphtheria toxin
in a composition can readily be determined using a flocculation
assay by comparison with a reference material calibrated against
such reference reagents. The conversion between IU and Lf systems
depends on the particular toxoid preparation.
[0019] In some embodiments of the invention a composition includes
a `low dose` of diphtheria toxoid. This means that the
concentration of diphtheria toxoid in the composition is .ltoreq.8
Lf/ml e.g. <7, <6, <5, <4 <3, <2, <1 Lf/ml,
etc. In a typical 0.5 ml unit dose volume, therefore, the amount of
diphtheria toxoid is less than 4 Lf e.g. <3, <2, <1,
<1/2 Lf, etc.
[0020] Where a composition of the invention includes an aluminium
salt adjuvant then diphtheria toxoid in the composition is
preferably adsorbed (more preferably totally adsorbed) onto that
salt, preferably onto an aluminium hydroxide adjuvant.
[0021] Tetanus Toxoid
[0022] Tetanus is caused by Clostridium tetani, a Gram-positive,
spore-forming bacillus. This organism expresses an endopeptidase
(`tetanus toxin`), which can be treated to give a toxoid that is no
longer toxic but that remains antigenic and is able to stimulate
the production of specific anti-toxin antibodies after injection.
Tetanus toxoids are disclosed in more detail in chapter 27 of
reference 1. Preferred tetanus toxoids are those prepared by
formaldehyde treatment. The tetanus toxoid can be obtained by
growing C. tetani in growth medium (e.g. a Latham medium derived
from bovine casein), followed by formaldehyde treatment,
ultrafiltration and precipitation. The material may then be treated
by a process comprising sterile filtration and/or dialysis.
[0023] Quantities of tetanus toxoid can be expressed in
international units (IU). For example, NIBSC supplies the `Tetanus
Toxoid Adsorbed Third International Standard 2000` [12,13], which
contains 469 IU per ampoule. As with diphtheria toxoid, the `Lf`
unit is an alternative to the IU system. NIBSC supplies `The 1st
International Reference Reagent for Tetanus Toxoid For Flocculation
Test` [14] which contains 1000 LF per ampoule. The concentration of
diphtheria toxin in a composition can readily be determined using a
flocculation assay by comparison with a reference material
calibrated against such reference reagents.
[0024] In some embodiments of the invention a composition includes
a `low dose` of tetanus toxoid. This means that the concentration
of tetanus toxoid in the composition is .ltoreq.3.5 Lf/ml e.g.
<3, <2.5, <2, <1.5<1, <1/2 Lf/ml, etc. In a
typical 0.5 ml unit dose volume, therefore, the amount of tetanus
toxoid is less than 1.75 Lf e.g. <1.5, <1, <1/2, <1/4
Lf, etc.
[0025] Where a composition of the invention includes an aluminium
salt adjuvant then tetanus toxoid in the composition is preferably
adsorbed (sometimes totally adsorbed) onto that salt, preferably
onto an aluminium hydroxide adjuvant.
[0026] Pertussis Toxoid
[0027] Bordetella pertussis causes whooping cough. Pertussis
antigens in vaccines are either cellular (whole cell, in the form
of inactivated B. pertussis cells; `wP`) or acellular (`aP`).
Preparation of cellular pertussis antigens is well documented (e.g.
see chapter 21 of reference 1) e.g. it may be obtained by heat
inactivation of phase I culture of B. pertussis. Where acellular
antigens are used, one, two or (preferably) three of the following
antigens are included: (1) detoxified pertussis toxin (pertussis
toxoid, or `PT`); (2) filamentous hemagglutinin (`FHA`); (3)
pertactin (also known as the `69 kiloDalton outer membrane
protein`). These three antigens can be prepared by isolation from
B. pertussis culture grown in modified Stainer-Scholte liquid
medium. PT and FHA can be isolated from the fermentation broth
(e.g. by adsorption on hydroxyapatite gel), whereas pertactin can
be extracted from the cells by heat treatment and flocculation
(e.g. using barium chloride). The antigens can be purified in
successive chromatographic and/or precipitation steps. PT and FHA
can be purified by hydrophobic chromatography, affinity
chromatography and size exclusion chromatography. Pertactin can be
purified by ion exchange chromatography, hydrophobic chromatography
and size exclusion chromatography, or by IMAC. FHA and pertactin
may be treated with formaldehyde prior to use according to the
invention. PT is preferably detoxified by treatment with
formaldehyde and/or glutaraldehyde. As an alternative to this
chemical detoxification procedure the PT may be a mutant PT in
which enzymatic activity has been reduced by mutagenesis [15] (e.g.
the 9K/129G double mutant [16]), but detoxification by chemical
treatment is preferred.
[0028] The invention can use a PT-containing wP antigen or,
preferably, a PT-containing aP antigen. When using an aP antigen a
composition of the invention will typically, in addition to the PT,
include FHA and, optionally, pertactin. It can also optionally
include fimbriae types 2 and 3.
[0029] Quantities of acellular pertussis antigens are typically
expressed in micrograms. In some embodiments of the invention a
composition includes a `low dose` of pertussis toxoid. This means
that the concentration of pertussis toxoid in the composition is
.ltoreq.5 .mu.g/ml e.g. <4, <3, <2.5, <2, <1
.mu.g/ml, etc. In a typical 0.5 ml unit dose volume, therefore, the
amount of pertussis toxoid is less than 2.5 .mu.g e.g. <2,
<1.5, <1, <0.5 .mu.g, etc.
[0030] Where a composition of the invention includes an aluminium
salt adjuvant then pertussis toxoid in the composition is
preferably adsorbed (sometimes totally adsorbed) onto that salt,
preferably onto an aluminium hydroxide adjuvant. Any FHA can also
be adsorbed to an aluminium hydroxide adjuvant. Any pertactin can
be adsorbed to an aluminium phosphate adjuvant.
[0031] Hib Conjugates
[0032] Haemophilus influenzae type b (`Hib`) causes bacterial
meningitis. Hib vaccines are typically based on the capsular
saccharide antigen (e.g. chapter 14 of ref. 1), the preparation of
which is well documented (e.g. references 17 to 26). The Hib
saccharide is conjugated to a carrier protein in order to enhance
its immunogenicity, especially in children. Typical carrier
proteins are tetanus toxoid, diphtheria toxoid, the CRM197
derivative of diphtheria toxoid, H. influenzae protein D, and an
outer membrane protein complex from serogroup B meningococcus.
Tetanus toxoid is a preferred carrier, as used in the product
commonly referred to as `PRP-T`. PRP-T can be made by activating a
Hib capsular polysaccharide using cyanogen bromide, coupling the
activated saccharide to an adipic acid linker (such as
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), typically the
hydrochloride salt), and then reacting the linker-saccharide entity
with a tetanus toxoid carrier protein. The saccharide moiety of the
conjugate may comprise full-length polyribosylribitol phosphate
(PRP) as prepared from Hib bacteria, and/or fragments of
full-length PRP. Conjugates with a saccharide:protein ratio (w/w)
of between 1:5 (i.e. excess protein) and 5:1 (i.e. excess
saccharide) may be used e.g. ratios between 1:2 and 5:1 and ratios
between 1:1.25 and 1:2.5. In preferred vaccines, however, the
weight ratio of saccharide to carrier protein is between 1:2.5 and
1:3.5. In vaccines where tetanus toxoid is present both as an
antigen and as a carrier protein then the weight ratio of
saccharide to carrier protein in the conjugate may be between 1:0.3
and 1:2 [27]. Administration of the Hib conjugate preferably
results in an anti-PRP antibody concentration of .gtoreq.0.15
.mu.g/ml, and more preferably .gtoreq.1 .mu.g/ml, and these are the
standard response thresholds.
[0033] Quantities of Hib antigens are typically expressed in
micrograms. For conjugate antigens this figure is based on the
saccharide content of the conjugate. In some embodiments of the
invention a composition includes a `low dose` of a Hib conjugate.
This means that the concentration of Hib saccharide in the
composition is .ltoreq.5 .mu.g/ml e.g. <4, <3, <2.5,
<2, <1, etc. In a typical 0.5 ml unit dose volume, therefore,
the amount of Hib is less than 2.5 .mu.g e.g. <2, <1.5,
<1, <0.5, etc.
[0034] Where a composition of the invention includes an aluminium
salt adjuvant then Hib conjugate can be adsorbed onto that salt or
can be unadsorbed.
[0035] Hepatitis B Virus Surface Antigen
[0036] Hepatitis B virus (HBV) is one of the known agents which
causes viral hepatitis. The HBV virion consists of an inner core
surrounded by an outer protein coat or capsid, and the viral core
contains the viral DNA genome. The major component of the capsid is
a protein known as HBV surface antigen or, more commonly, `HBsAg`,
which is typically a 226-amino acid polypeptide with a molecular
weight of .about.24 kDa. All existing hepatitis B vaccines contain
HBsAg, and when this antigen is administered to a normal vaccinee
it stimulates the production of anti-HBsAg antibodies which protect
against HBV infection.
[0037] For vaccine manufacture, HBsAg can be made in two ways. The
first method involves purifying the antigen in particulate form
from the plasma of chronic hepatitis B carriers, as large
quantities of HBsAg are synthesized in the liver and released into
the blood stream during an HBV infection. The second way involves
expressing the protein by recombinant DNA methods. HBsAg for use
with the method of the invention is recombinantly expressed in
yeast cells. Suitable yeasts include Saccharomyces (such as S.
cerevisiae) or Hanensula (such as H. polymorpha) hosts.
[0038] Unlike native HBsAg (i.e. as in the plasma-purified
product), yeast-expressed HBsAg is generally non-glycosylated, and
this is the most preferred form of HBsAg for use with the
invention. Yeast-expressed HBsAg is highly immunogenic and can be
prepared without the risk of blood product contamination.
[0039] The HBsAg will generally be in the form of
substantially-spherical particles (average diameter of about 20
nm), including a lipid matrix comprising phospholipids.
Yeast-expressed HBsAg particles may include phosphatidylinositol,
which is not found in natural HBV virions. The particles may also
include a non-toxic amount of LPS in order to stimulate the immune
system [28]. The particles may retain non-ionic surfactant (e.g.
polysorbate 20) if this was used during disruption of yeast
[29].
[0040] A preferred method for HBsAg purification involves, after
cell disruption: ultrafiltration; size exclusion chromatography;
anion exchange chromatography; ultracentrifugation; desalting; and
sterile filtration. Lysates may be precipitated after cell
disruption (e.g. using a polyethylene glycol), leaving HBsAg in
solution, ready for ultrafiltration.
[0041] After purification HBsAg may be subjected to dialysis (e.g.
with cysteine), which can be used to remove any mercurial
preservatives such as thimerosal that may have been used during
HBsAg preparation [30]. Thimerosal-free preparation is
preferred.
[0042] The HBsAg is preferably from HBV subtype adw2.
[0043] Quantities of HBsAg are typically expressed in micrograms.
In some embodiments of the invention a composition includes a `low
dose` of HBsAg. This means that the concentration of HBsAg in the
composition is .ltoreq.5 .mu.g/ml e.g. <4, <3, <2.5,
<2, <1, etc. In a typical 0.5 ml unit dose volume, therefore,
the amount of HBsAg is less than 2.5 .mu.g e.g. <2, <1.5,
<1, <0.5, etc.
[0044] Where a composition of the invention includes an aluminium
salt adjuvant then HBsAg can be adsorbed onto that salt (preferably
adsorbed onto an aluminium phosphate adjuvant).
[0045] Inactivated Poliovirus Antigen (IPV)
[0046] Poliomyelitis can be caused by one of three types of
poliovirus. The three types are similar and cause identical
symptoms, but they are antigenically very different and infection
by one type does not protect against infection by others. As
explained in chapter 24 of reference 1, it is therefore preferred
to use three poliovirus antigens with the invention--poliovirus
Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-1
strain), and poliovirus Type 3 (e.g. Saukett strain). As an
alternative to these strains, Sabin strains of types 1 to 3 can be
used e.g. as discussed in references 31 & 32.
[0047] Polioviruses may be grown in cell culture. A preferred
culture uses a Vero cell line, which is a continuous cell line
derived from monkey kidney. Vero cells can conveniently be cultured
microcarriers. Culture of the Vero cells before and during viral
infection may involve the use of bovine-derived material, such as
calf serum, and of lactalbumin hydrolysate (e.g. obtained by
enzymatic degradation of lactalbumin). Such bovine-derived material
should be obtained from sources which are free from BSE or other
TSEs.
[0048] After growth, virions may be purified using techniques such
as ultrafiltration, diafiltration, and chromatography. Prior to
administration to patients, polioviruses must be inactivated, and
this can be achieved by treatment with formaldehyde before the
viruses are used in the process of the invention.
[0049] The viruses are preferably grown, purified and inactivated
individually, and are then combined to give a bulk mixture for use
with the invention.
[0050] Quantities of inactivated poliovirus (IPV) are typically
expressed in the `DU` unit (the "D-antigen unit" [33]). In some
embodiments of the invention a composition includes a low dose' of
a poliovirus. For a Type 1 poliovirus this means that the
concentration of the virus in the composition is .ltoreq.20 DU/ml
e.g. <18, <16, <14, <12, <10, etc. For a Type 2
poliovirus this means that the concentration of the virus in the
composition is .ltoreq.4 DU/ml e.g. <3, <2, <1, <0.5,
etc. For a Type 3 poliovirus this means that the concentration of
the virus in the composition is .ltoreq.16 DU/ml e.g. <14,
<12, <10, <8, <6, etc. Where all three of Types 1, 2
and 3 poliovirus are present the three antigens can be present at a
DU ratio of 5:1:4 respectively, or at any other suitable ratio e.g.
a ratio of 15:32:45 when using Sabin strains [31]. A low dose of
antigen from Sabin strains is particularly useful, with .ltoreq.10
DU type 1, .ltoreq.20 DU type 2, and .ltoreq.30 DU type 3 (per unit
dose).
[0051] Where a composition of the invention includes an aluminium
salt adjuvant then polioviruses are preferably not adsorbed to any
adjuvant before they are formulated, but after formulation they may
become adsorbed onto any aluminium adjuvant(s) in the
composition.
[0052] Further Antigens
[0053] As well as including D, T, Pa, HBsAg, Hib and/or poliovirus
antigens, immunogenic compositions of the invention may include
antigens from further pathogens. For example, these antigens may be
from N. meningitidis (one or more of serogroups A, B, C, W135
and/or Y) or S. pneumoniae.
[0054] Meningococcal Saccharides
[0055] Where a composition includes a Neisseria meningitidis
capsular saccharide conjugate there may be one or more than one
such conjugate. Including 2, 3, or 4 of serogroups A, C, W135 and Y
is typical e.g. A+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135,
A+C+Y, A+W135+Y, A+C+W135+Y, etc. Components including saccharides
from all four of serogroups A, C, W135 and Y are useful, as in the
MENACTRA.TM. and MENVEO.TM. products. Where conjugates from more
than one serogroup are included then they may be present at
substantially equal masses e.g. the mass of each serogroup's
saccharide is within .+-.10% of each other. A typical quantity per
serogroup is between 1 .mu.g and 20 .mu.g e.g. between 2 and 10
.mu.g per serogroup, or about 4 .mu.g or about 5 .mu.g or about 10
.mu.g. As an alternative to a substantially equal ratio, a double
mass of serogroup A saccharide may be used.
[0056] Administration of a conjugate preferably results in an
increase in serum bactericidal assay (SBA) titre for the relevant
serogroup of at least 4-fold, and preferably at least 8-fold. SBA
titres can be measured using baby rabbit complement or human
complement [34].
[0057] The capsular saccharide of serogroup A meningococcus is a
homopolymer of (.alpha.1.fwdarw.6)-linked
N-acetyl-D-mannosamine-1-phosphate, with partial 0-acetylation in
the C3 and C4 positions. Acetylation at the C-3 position can be
70-95%. Conditions used to purify the saccharide can result in
de-O-acetylation (e.g. under basic conditions), but it is useful to
retain OAc at this C-3 position. In some embodiments, at least 50%
(e.g. at least 60%, 70%, 80%, 90%, 95% or more) of the mannosamine
residues in a serogroup A saccharides are O-acetylated at the C-3
position. Acetyl groups can be replaced with blocking groups to
prevent hydrolysis [35], and such modified saccharides are still
serogroup A saccharides within the meaning of the invention.
[0058] The serogroup C capsular saccharide is a homopolymer of
(.alpha.2.fwdarw.9)-linked sialic acid (N-acetyl neuraminic acid,
or `NeuNAc`). The saccharide structure is written as .fwdarw.9)-Neu
p NAc 7/8 OAc-(.alpha.2.fwdarw.. Most serogroup C strains have
0-acetyl groups at C-7 and/or C-8 of the sialic acid residues, but
about 15% of clinical isolates lack these 0-acetyl groups [36,37].
The presence or absence of OAc groups generates unique epitopes,
and the specificity of antibody binding to the saccharide may
affect its bactericidal activity against O-acetylated (OAc-) and
de-O-acetylated (OAc+) strains [38-40]. Serogroup C saccharides
used with the invention may be prepared from either OAc+ or OAc-
strains. Licensed MenC conjugate vaccines include both OAc-
(NEISVAC-C.TM.) and OAc+ (MENJUGATE.TM. & MENINGITEC.TM.)
saccharides. In some embodiments, strains for production of
serogroup C conjugates are OAc+ strains, e.g. of serotype 16,
serosubtype P1.7a, 1, etc. Thus C:16:P1.7a,1 OAc+ strains may be
used. OAc+ strains in serosubtype P1.1 are also useful, such as the
C11 strain. Preferred MenC saccharides are taken from OAc+ strains,
such as strain C11.
[0059] The serogroup W135 saccharide is a polymer of sialic
acid-galactose disaccharide units. Like the serogroup C saccharide,
it has variable 0-acetylation, but at sialic acid 7 and 9 positions
[41]. The structure is written as:
.fwdarw.4)-D-Neup5Ac(7/9OAc)-.alpha.-(2.fwdarw.6)-D-Gal-.alpha.-(1.fwdarw-
..
[0060] The serogroup Y saccharide is similar to the serogroup W135
saccharide, except that the disaccharide repeating unit includes
glucose instead of galactose. Like serogroup W135, it has variable
0-acetylation at sialic acid 7 and 9 positions [41]. The serogroup
Y structure is written as:
.fwdarw.4)-D-Neup5Ac(7/9OAc)-.alpha.-(2-6)-D-Glc-.alpha.-(1.fwdarw..
[0061] The saccharides used according to the invention may be
O-acetylated as described above (e.g. with the same 0-acetylation
pattern as seen in native capsular saccharides), or they may be
partially or totally de-O-acetylated at one or more positions of
the saccharide rings, or they may be hyper-O-acetylated relative to
the native capsular saccharides. For example, reference 42 reports
the use of serogroup Y saccharides that are more than 80%
de-O-acetylated.
[0062] The saccharide moieties in meningococcal conjugates may
comprise full-length saccharides as prepared from meningococci,
and/or may comprise fragments of full-length saccharides i.e. the
saccharides may be shorter than the native capsular saccharides
seen in bacteria. The saccharides may thus be depolymerised, with
depolymerisation occurring during or after saccharide purification
but before conjugation. Depolymerisation reduces the chain length
of the saccharides. One depolymerisation method involves the use of
hydrogen peroxide [43]. Hydrogen peroxide is added to a saccharide
(e.g. to give a final H.sub.2O.sub.2 concentration of 1%), and the
mixture is then incubated (e.g. at about 55.degree. C.) until a
desired chain length reduction has been achieved. Another
depolymerisation method involves acid hydrolysis [44]. Other
depolymerisation methods are known in the art. The saccharides used
to prepare conjugates for use according to the invention may be
obtainable by any of these depolymerisation methods.
Depolymerisation can be used in order to provide an optimum chain
length for immunogenicity and/or to reduce chain length for
physical manageability of the saccharides. In some embodiments,
saccharides have the following range of average degrees of
polymerisation (Dp): A=10-20; C=12-22; W135=15-25; Y=15-25. In
terms of molecular weight, rather than Dp, useful ranges are, for
all serogroups: <100 kDa; 5 kDa-75 kDa; 7 kDa-50 kDa; 8 kDa-35
kDa; 12 kDa-25 kDa; 15 kDa-22 kDa. In other embodiments, the
average molecular weight for saccharides from each of meningococcal
serogroups A, C, W135 and Y may be more than 50 kDa e.g. .gtoreq.75
kDa, .gtoreq.100 kDa, .gtoreq.110 kDa, .gtoreq.120 kDa, .gtoreq.130
kDa, etc. [45], and even up to 1500 kDa, in particular as
determined by MALLS. For instance: a MenA saccharide may be in the
range 50-500 kDa e.g. 60-80 kDa; a MenC saccharide may be in the
range 100-210 kDa; a MenW135 saccharide may be in the range 60-190
kDa e.g. 120-140 kDa; and/or a MenY saccharide may be in the range
60-190 kDa e.g. 150-160 kDa.
[0063] If a component or composition includes both Hib and
meningococcal conjugates then, in some embodiments, the mass of Hib
saccharide can be substantially the same as the mass of a
particular meningococcal serogroup saccharide. In some embodiments,
the mass of Hib saccharide will be more than (e.g. at least
1.5.times.) the mass of a particular meningococcal serogroup
saccharide. In some embodiments, the mass of Hib saccharide will be
less than (e.g. at least 1.5.times. less) the mass of a particular
meningococcal serogroup saccharide.
[0064] Where a composition includes saccharide from more than one
meningococcal serogroup, there is an mean saccharide mass per
serogroup. If substantially equal masses of each serogroup are used
then the mean mass will be the same as each individual mass; where
non-equal masses are used then the mean will differ e.g. with a
10:5:5:5 .mu.g amount for a MenACWY mixture, the mean mass is 6.25
.mu.g per serogroup. In some embodiments, the mass of Hib
saccharide will be substantially the same as the mean mass of
meningococcal saccharide per serogroup. In some embodiments, the
mass of Hib saccharide will be more than (e.g. at least 1.5.times.)
the mean mass of meningococcal saccharide per serogroup. In some
embodiments, the mass of Hib saccharide will be less than (e.g. at
least 1.5.times.) the mean mass of meningococcal saccharide per
serogroup [46].
[0065] Meningococcal Polypeptides
[0066] The capsular saccharide of Neisseria meningitidis serogroup
B is not a useful vaccine immunogen and so polypeptide antigens can
be used instead. For instance, the "universal vaccine for serogroup
B meningococcus" reported by Novartis Vaccines in ref. 47 can be
used with the invention.
[0067] A composition of the invention can include a factor H
binding protein (fHBP) antigen. The fHBP antigen has been
characterised in detail. It has also been known as protein `741`
[SEQ IDs 2535 & 2536 in ref. 48], `NMB1870`, `GNA1870` [refs.
49-51], `P2086`, `LP2086` or `ORF2086` [52-54]. It is naturally a
lipoprotein and is expressed across all meningococcal serogroups.
The fHBP antigen falls into three distinct variants [55] and it is
preferred to include antigens for all variants.
[0068] A composition of the invention may include a Neisserial
Heparin Binding Antigen (NHBA) [56]. This antigen was included in
the published genome sequence for meningococcal serogroup B strain
MC58 [57] as gene NMB2132.
[0069] A composition of the invention may include a NadA antigen.
The NadA antigen was included in the published genome sequence for
meningococcal serogroup B strain MC58 [57] as gene NMB1994.
[0070] A composition of the invention may include a NspA antigen.
The NspA antigen was included in the published genome sequence for
meningococcal serogroup B strain MC58 [57] as gene NMB0663.
[0071] A composition of the invention may include a NhhA antigen.
The NhhA antigen was included in the published genome sequence for
meningococcal serogroup B strain MC58 [57] as gene NMB0992.
[0072] A composition of the invention may include an App antigen.
The App antigen was included in the published genome sequence for
meningococcal serogroup B strain MC58 [57] as gene NMB1985.
[0073] A composition of the invention may include an Omp85 antigen.
Omp85 was included in the published genome sequence for
meningococcal serogroup B strain MC58 [57] as gene NMB0182.
[0074] A composition of the invention may include a meningococcal
outer membrane vesicle.
[0075] Pneumococcal Saccharides
[0076] Streptococcus pneumoniae causes bacterial meningitis and
existing vaccines are based on capsular saccharides. Thus
compositions of the invention can include at least one pneumococcal
capsular saccharide conjugated to a carrier protein.
[0077] The invention can include capsular saccharide from one or
more different pneumococcal serotypes. Where a composition includes
saccharide antigens from more than one serotype, these are
preferably prepared separately, conjugated separately, and then
combined. Methods for purifying pneumococcal capsular saccharides
are known in the art (e.g. see reference 58) and vaccines based on
purified saccharides from 23 different serotypes have been known
for many years. Improvements to these methods have also been
described e.g. for serotype 3 as described in reference 59, or for
serotypes 1, 4, 5, 6A, 6B, 7F and 19A as described in reference
60.
[0078] Pneumococcal capsular saccharide(s) will typically be
selected from the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F,
8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F
and/or 33F. Thus, in total, a composition may include a capsular
saccharide from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23 or more different serotypes.
Compositions which include at least serotype 6B saccharide are
useful.
[0079] A useful combination of serotypes is a 7-valent combination
e.g. including capsular saccharide from each of serotypes 4, 6B,
9V, 14, 18C, 19F, and 23F. Another useful combination is a 9-valent
combination e.g. including capsular saccharide from each of
serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. Another useful
combination is a 10-valent combination e.g. including capsular
saccharide from each of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F
and 23F. An 11-valent combination may further include saccharide
from serotype 3. A 12-valent combination may add to the 10-valent
mixture: serotypes 6A and 19A; 6A and 22F; 19A and 22F; 6A and 15B;
19A and 15B; or 22F and 15B. A 13-valent combination may add to the
11-valent mixture: serotypes 19A and 22F; 8 and 12F; 8 and 15B; 8
and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12F and 22F; 15B and
19A; 15B and 22F; 6A and 19A, etc.
[0080] Thus a useful 13-valent combination includes capsular
saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19
(or 19A), 19F and 23F e.g. prepared as disclosed in references 61
to 64. One such combination includes serotype 6B saccharide at
about 8 .mu.g/ml and the other 12 saccharides at concentrations of
about 4 .mu.g/ml each. Another such combination includes serotype
6A and 6B saccharides at about 8 .mu.g/ml each and the other 11
saccharides at about 4 .mu.g/ml each.
[0081] Suitable carrier proteins for conjugates include bacterial
toxins, such as diphtheria or tetanus toxins, or toxoids or mutants
thereof. These are commonly used in conjugate vaccines. For
example, the CRM197 diphtheria toxin mutant is useful [65]. Other
suitable carrier proteins include synthetic peptides [66,67], heat
shock proteins [68,69], pertussis proteins [70,71], cytokines [72],
lymphokines [72], hormones [72], growth factors [72], artificial
proteins comprising multiple human CD4.sup.+ T cell epitopes from
various pathogen-derived antigens [73] such as N19 [74], protein D
from H. influenzae [75-77], pneumolysin [78] or its non-toxic
derivatives [79], pneumococcal surface protein PspA [80],
iron-uptake proteins [81], toxin A or B from C. difficile [82],
recombinant Pseudomonas aeruginosa exoprotein A (rEPA) [83],
etc.
[0082] Particularly useful carrier proteins for pneumococcal
conjugate vaccines are CRM197, tetanus toxoid, diphtheria toxoid
and H. influenzae protein D. CRM197 is used in PREVNAR.TM.. A
13-valent mixture may use CRM197 as the carrier protein for each of
the 13 conjugates, and CRM197 may be present at about 55-60
.mu.g/ml.
[0083] Where a composition includes conjugates from more than one
pneumococcal serotype, it is possible to use the same carrier
protein for each separate conjugate, or to use different carrier
proteins. In both cases, though, a mixture of different conjugates
will usually be formed by preparing each serotype conjugate
separately, and then mixing them to form a mixture of separate
conjugates. Reference 84 describes potential advantages when using
different carrier proteins in multivalent pneumococcal conjugate
vaccines, but the PREVNAR.TM. product successfully uses the same
carrier for each of seven different serotypes.
[0084] A carrier protein may be covalently conjugated to a
pneumococcal saccharide directly or via a linker. Various linkers
are known. For example, attachment may be via a carbonyl, which may
be formed by reaction of a free hydroxyl group of a modified
saccharide with CDI [85,86] followed by reaction with a protein to
form a carbamate linkage. Carbodiimide condensation can be used
[87]. An adipic acid linker can be used, which may be formed by
coupling a free --NH.sub.2 group (e.g. introduced to a saccharide
by amination) with adipic acid (using, for example, diimide
activation), and then coupling a protein to the resulting
saccharide-adipic acid intermediate [88,89]. Other linkers include
.beta.-propionamido [90], nitrophenyl-ethylamine [91], haloacyl
halides [92], glycosidic linkages [93], 6-aminocaproic acid [94],
N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [95], adipic
acid dihydrazide ADH [96], C.sub.4 to C.sub.12 moieties [97],
etc.
[0085] Conjugation via reductive amination can be used. The
saccharide may first be oxidised with periodate to introduce an
aldehyde group, which can then form a direct covalent linkage to a
carrier protein via reductive amination e.g. to the .epsilon.-amino
group of a lysine. If the saccharide includes multiple aldehyde
groups per molecule then this linkage technique can lead to a
cross-linked product, where multiple aldehydes react with multiple
carrier amines. This cross-linking conjugation technique is
particularly useful for at least pneumococcal serotypes 4, 6B, 9V,
14, 18C, 19F and 23F.
[0086] A pneumococcal saccharide may comprise a full-length intact
saccharide as prepared from pneumococcus, and/or may comprise
fragments of full-length saccharides i.e. the saccharides may be
shorter than the native capsular saccharides seen in bacteria. The
saccharides may thus be depolymerised, with depolymerisation
occurring during or after saccharide purification but before
conjugation. Depolymerisation reduces the chain length of the
saccharides. Depolymerisation can be used in order to provide an
optimum chain length for immunogenicity and/or to reduce chain
length for physical manageability of the saccharides. Where more
than one pneumococcal serotype is used then it is possible to use
intact saccharides for each serotype, fragments for each serotype,
or to use intact saccharides for some serotypes and fragments for
other serotypes.
[0087] Where a composition includes saccharide from any of
serotypes 4, 6B, 9V, 14, 19F and 23F, these saccharides are
preferably intact. In contrast, where a composition includes
saccharide from serotype 18C, this saccharide is preferably
depolymerised.
[0088] A serotype 3 saccharide may also be depolymerised, For
instance, a serotype 3 saccharide can be subjected to acid
hydrolysis for depolymerisation [61] e.g. using acetic acid. The
resulting fragments may then be oxidised for activation (e.g.
periodate oxidation, maybe in the presence of bivalent cations e.g.
with MgCl.sub.2), conjugated to a carrier (e.g. CRM197) under
reducing conditions (e.g. using sodium cyanoborohydride), and then
(optionally) any unreacted aldehydes in the saccharide can be
capped (e.g. using sodium borohydride) [61]. Conjugation may be
performed on lyophilized material e.g. after co-lyophilizing
activated saccharide and carrier.
[0089] A serotype 1 saccharide may be at least partially
de-O-acetylated e.g. achieved by alkaline pH buffer treatment [62]
such as by using a bicarbonate/carbonate buffer. Such (partially)
de-O-acetylated saccharides can be oxidised for activation (e.g.
periodate oxidation), conjugated to a carrier (e.g. CRM197) under
reducing conditions (e.g. using sodium cyanoborohydride), and then
(optionally) any unreacted aldehydes in the saccharide can be
capped (e.g. using sodium borohydride) [62]. Conjugation may be
performed on lyophilized material e.g. after co-lyophilizing
activated saccharide and carrier.
[0090] A serotype 19A saccharide may be oxidised for activation
(e.g. periodate oxidation), conjugated to a carrier (e.g. CRM197)
in DMSO under reducing conditions, and then (optionally) any
unreacted aldehydes in the saccharide can be capped (e.g. using
sodium borohydride) [98]. Conjugation may be performed on
lyophilized material e.g. after co-lyophilizing activated
saccharide and carrier.
[0091] One or more pneumococcal capsular saccharide conjugates may
be present in lyophilised form.
[0092] Pneumococcal conjugates can ideally elicit anticapsular
antibodies that bind to the relevant saccharide e.g. elicit an
anti-saccharide antibody level .gtoreq.0.20 .mu.g/mL [99]. The
antibodies may be evaluated by enzyme immunoassay (EIA) and/or
measurement of opsonophagocytic activity (OPA). The EIA method has
been extensively validated and there is a link between antibody
concentration and vaccine efficacy.
[0093] Aluminium Salt Adjuvants
[0094] In some embodiments, compositions of the invention include
an aluminium salt adjuvant, although other embodiments may be
aluminium-free.
[0095] Aluminium salt adjuvants currently in use are typically
referred to either as "aluminium hydroxide" or as "aluminium
phosphate" adjuvants. These are names of convenience, however, as
neither is a precise description of the actual chemical compound
which is present (e.g. see chapter 9 of reference 100). The
invention can use any of the "hydroxide" or "phosphate" salts that
useful as adjuvants.
[0096] Aluminium salts which include hydroxide ions are the
preferred insoluble metal salts for use with the present invention
as these hydroxide ions can readily undergo ligand exchange for
adsorption of antigen and/or TLR agonists. Thus preferred salts for
adsorption of TLR agonists are aluminium hydroxide and/or aluminium
hydroxyphosphate. These have surface hydroxyl moieties which can
readily undergo ligand exchange with phosphorus-containing groups
(e.g. phosphates, phosphonates) to provide stable adsorption. An
aluminium hydroxide adjuvant is most preferred.
[0097] The adjuvants known as "aluminium hydroxide" are typically
aluminium oxyhydroxide salts, which are usually at least partially
crystalline. Aluminium oxyhydroxide, which can be represented by
the formula AlO(OH), can be distinguished from other aluminium
compounds, such as aluminium hydroxide Al(OH).sub.3, by infrared
(IR) spectroscopy, in particular by the presence of an adsorption
band at 1070 cm.sup.-1 and a strong shoulder at 3090-3100 cm.sup.-1
(chapter 9 of ref. 100). The degree of crystallinity of an
aluminium hydroxide adjuvant is reflected by the width of the
diffraction band at half height (WHH), with poorly-crystalline
particles showing greater line broadening due to smaller
crystallite sizes. The surface area increases as WHH increases, and
adjuvants with higher WHH values have been seen to have greater
capacity for antigen adsorption. A fibrous morphology (e.g. as seen
in transmission electron micrographs) is typical for aluminium
hydroxide adjuvants e.g. with needle-like particles with diameters
about 2 nm. The PZC of aluminium hydroxide adjuvants is typically
about 11 i.e. the adjuvant itself has a positive surface charge at
physiological pH. Adsorptive capacities of between 1.8-2.6 mg
protein per mg Al.sup.+++ at pH 7.4 have been reported for
aluminium hydroxide adjuvants.
[0098] The adjuvants known as "aluminium phosphate" are typically
aluminium hydroxyphosphates, often also containing a small amount
of sulfate. They may be obtained by precipitation, and the reaction
conditions and concentrations during precipitation influence the
degree of substitution of phosphate for hydroxyl in the salt.
Hydroxyphosphates generally have a PO.sub.4/Al molar ratio between
0.3 and 0.99. Hydroxyphosphates can be distinguished from strict
AlPO.sub.4 by the presence of hydroxyl groups. For example, an IR
spectrum band at 3164 cm.sup.-1 (e.g. when heated to 200.degree.
C.) indicates the presence of structural hydroxyls (chapter 9 of
ref. 100).
[0099] The PO.sub.4/Al.sup.3+ molar ratio of an aluminium phosphate
adjuvant will generally be between 0.3 and 1.2, preferably between
0.8 and 1.2, and more preferably 0.95.+-.0.1. The aluminium
phosphate will generally be amorphous, particularly for
hydroxyphosphate salts. A typical adjuvant is amorphous aluminium
hydroxyphosphate with PO.sub.4/Al molar ratio between 0.84 and
0.92, included at 0.6 mg Al.sup.3+/ml. The aluminium phosphate will
generally be particulate. Typical diameters of the particles are in
the range 0.5-20 .mu.m (e.g. about 5-10 .mu.m) after any antigen
adsorption. Adsorptive capacities of between 0.7-1.5 mg protein per
mg Al.sup.+++ at pH 7.4 have been reported for aluminium phosphate
adjuvants.
[0100] The PZC of aluminium phosphate is inversely related to the
degree of substitution of phosphate for hydroxyl, and this degree
of substitution can vary depending on reaction conditions and
concentration of reactants used for preparing the salt by
precipitation. PZC is also altered by changing the concentration of
free phosphate ions in solution (more phosphate=more acidic PZC) or
by adding a buffer such as a histidine buffer (makes PZC more
basic). Aluminium phosphates used according to the invention will
generally have a PZC of between 4.0 and 7.0, more preferably
between 5.0 and 6.5 e.g. about 5.7.
[0101] In solution both aluminium phosphate and hydroxide adjuvants
tend to form stable porous aggregates 1-10 .mu.m in diameter
[101].
[0102] A composition can include a mixture of both an aluminium
hydroxide and an aluminium phosphate, and components may be
adsorbed to one or both of these salts.
[0103] An aluminium phosphate solution used to prepare a
composition of the invention may contain a buffer (e.g. a phosphate
or a histidine or a Tris buffer), but this is not always necessary.
The aluminium phosphate solution is preferably sterile and
pyrogen-free. The aluminium phosphate solution may include free
aqueous phosphate ions e.g. present at a concentration between 1.0
and 20 mM, preferably between 5 and 15 mM, and more preferably
about 10 mM. The aluminium phosphate solution may also comprise
sodium chloride. The concentration of sodium chloride is preferably
in the range of 0.1 to 100 mg/ml (e.g. 0.5-50 mg/ml, 1-20 mg/ml,
2-10 mg/ml) and is more preferably about 3.+-.1 mg/ml. The presence
of NaCl facilitates the correct measurement of pH prior to
adsorption of antigens.
[0104] In some embodiments of the invention a composition includes
less than 0.2 mg Al.sup.+++ per unit dose. The amount of Al.sup.+++
can be lower than this e.g. <150 .mu.g, <100 .mu.g, <75
.mu.g, <50 .mu.g, <25 .mu.g, <10 .mu.g, etc.
[0105] In some embodiments of the invention a composition has an
Al.sup.+++ concentration below 0.4 mg/ml. The concentration of
Al.sup.+++ can be lower than this e.g. <300 .mu.g/ml, <250
.mu.g/ml, <200 .mu.g/ml, <150 .mu.g/ml, <100 .mu.g/ml,
<75 .mu.g/ml, <50 .mu.g/ml, <20 .mu.g/ml, etc.
[0106] Where compositions of the invention include an
aluminium-based adjuvant, settling of components may occur during
storage. The composition should therefore be shaken prior to
administration to a patient. The shaken composition will be a
turbid white suspension.
[0107] Toll-Like Receptor Agonists
[0108] Where a composition of the invention includes an aluminium
salt adjuvant then it is possible to adsorb a TLR agonist to that
aluminium salt, thereby improving the immunopotentiating effect of
the adjuvant [102]. This can lead to a better immune response
and/or permits a reduction in the amount of aluminium in the
composition while maintaining an equivalent adjuvant effect.
[0109] A composition of the invention can therefore include an
aluminium salt (preferably an aluminium hydroxide) to which a TLR
agonist (preferably a TLR7 agonist, and more preferably an agonist
of human TLR7) is adsorbed. The agonist and the salt can form a
stable adjuvant complex which retains the salt's ability to adsorb
antigens.
[0110] TLR agonists with adsorptive properties typically include a
phosphorus-containing moiety which can undergo ligand exchange with
surface groups on an aluminium salt e.g. with surface hydroxide
groups. Thus a useful TLR agonist may include a phosphate, a
phosphonate, a phosphinate, a phosphonite, a phosphinite, a
phosphate, etc. Preferred TLR agonists include at least one
phosphate or phosphonate group [102].
[0111] Useful adsorptive TLR2 and TLR7 agonists are disclosed in
references 102 to 106. Specific adsorptive TLR7 agonists of
interest include, but are not limited to, compounds 1A to 27A in
Table A on pages 79-84 of reference 107. For instance, the TLR7
agonist can be one of:
TABLE-US-00002 ##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027## ##STR00028##
[0112] These compounds can be adsorbed to aluminium salt adjuvants
by simple mixing. For instance, the compound (1 mg/mL) can be
dissolved in 10 mM NaOH and added to a suspension of aluminium
hydroxide adjuvant (2 mg/mL) to give a final TLR agonist
concentration of 100 .mu.g/dose. Preferably, 0.1 mg/mL, more
preferably 0.01 mg/mL of the compound is added to 2 mg/mL aluminium
hydroxide. The mass ratio of aluminium salt to TLR agonist is
between 2:1 and 400:1, preferably 20:1, more preferably 200:1.
Incubation at room temperature for 1 hour usually suffices for
>90% adsorption. Adsorption can take place across a range of pH,
e.g. from 6.5 to 9. In a preferred embodiment, an aluminium salt
and a TLR agonist are prepared in histidine buffer e.g. between
5-20 mM (such as 10 mM) histidine buffer, conveniently at pH 6.5.
For optimal antigen adsorption on aluminium hydroxide, the pH
should be in the range between 6.0 and 6.5. The pH is also crucial
for the integrity and stability of the antigens, and in case of
protein antigens, for their proper folding in the final vaccine
formulation.
[0113] One useful TLR7 agonist, which is used in the examples
below, is `compound T` (compound 6A on page 80 of reference 107).
It has a solubility of about 4 mg/ml in water and adsorbs well to
aluminium hydroxide:
##STR00029##
[0114] In general, when a composition includes both a TLR agonist
and an aluminium salt, the weight ratio of agonist to Al.sup.+++
will be less than 5:1 e.g. less than 4:1, less than 3:1, less than
2:1, or less than 1:1. Thus, for example, with an Al.sup.+++
concentration of 0.5 mg/ml the maximum concentration of TLR agonist
would be 2.5 mg/ml. But higher or lower levels can be used. A lower
mass of TLR agonist than of Al.sup.+++ is typical e.g. per dose,
100 .mu.g of TLR agonist with 0.2 mg Al.sup.+++, etc.
[0115] The amount of TLR agonist in a unit dose will fall in a
relatively broad range that can be determined through routine
trials. An amount of between 1-1000 .mu.g/dose can be used e.g.
from 5-100 .mu.g per dose or from 10-100 .mu.g per dose, and
ideally .ltoreq.300 .mu.g per dose e.g. about 5 .mu.g, 10 .mu.g, 20
.mu.g, 25 .mu.g, 50 .mu.g or 100 .mu.g per dose. Thus the
concentration of a TLR agonist in a composition of the invention
may be from 2-2000 .mu.g/ml e.g. from 10-200 .mu.g/ml, or about 5,
10, 20, 40, 50, 100 or 200 .mu.g/ml, and ideally .ltoreq.600
.mu.g/ml.
[0116] It is preferred that at least 50% (by mass) of an agonist in
the composition is adsorbed to the metal salt e.g. .gtoreq.60%,
.gtoreq.70%, .gtoreq.80%, .gtoreq.85%, .gtoreq.90%, .gtoreq.92%,
.gtoreq.94%, .gtoreq.95%, .gtoreq.96%, .gtoreq.97%, .gtoreq.98%,
.gtoreq.99%, or even 100%.
[0117] Where a composition of the invention includes a TLR agonist
adsorbed to a metal salt, and also includes a buffer, it is
preferred that the concentration of any phosphate ions in the
buffer should be less than 50 mM (e.g. between 1-15 mM) as a high
concentration of phosphate ions can cause desorption. Use of a
histidine buffer is preferred.
[0118] Oil-in-Water Emulsion Adjuvants
[0119] Oil-in-water emulsions are known to be useful adjuvants e.g.
MF59 and AS03 are both present in authorised vaccines in Europe.
Various useful emulsion adjuvants are known, and they typically
include at least one oil and at least one surfactant, with the
oil(s) and surfactant(s) being biodegradable (metabolisable) and
biocompatible. The oil droplets in the emulsion generally have a
sub-micron diameter, with these small sizes being achieved with a
microfluidiser to provide stable emulsions. Droplets with a size
less than 220 nm are preferred as they can be subjected to filter
sterilization.
[0120] The invention can be used with oils such as those from an
animal (such as fish) or vegetable source. Sources for vegetable
oils include nuts, seeds and grains. Peanut oil, soybean oil,
coconut oil, and olive oil, the most commonly available, exemplify
the nut oils. Jojoba oil can be used e.g. obtained from the jojoba
bean. Seed oils include safflower oil, cottonseed oil, sunflower
seed oil, sesame seed oil and the like. In the grain group, corn
oil is the most readily available, but the oil of other cereal
grains such as wheat, oats, rye, rice, teff, triticale and the like
may also be used. 6-10 carbon fatty acid esters of glycerol and
1,2-propanediol, while not occurring naturally in seed oils, may be
prepared by hydrolysis, separation and esterification of the
appropriate materials starting from the nut and seed oils. Fats and
oils from mammalian milk are metabolizable and may therefore be
used in the practice of this invention. The procedures for
separation, purification, saponification and other means necessary
for obtaining pure oils from animal sources are well known in the
art. Most fish contain metabolizable oils which may be readily
recovered. For example, cod liver oil, shark liver oils, and whale
oil such as spermaceti exemplify several of the fish oils which may
be used herein. A number of branched chain oils are synthesized
biochemically in 5-carbon isoprene units and are generally referred
to as terpenoids. Shark liver oil contains a branched, unsaturated
terpenoids known as squalene,
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which
is particularly preferred herein. Squalane, the saturated analog to
squalene, is also a preferred oil. Fish oils, including squalene
and squalane, are readily available from commercial sources or may
be obtained by methods known in the art. Other preferred oils are
the tocopherols (see below). Mixtures of oils can be used.
[0121] Surfactants can be classified by their `HLB`
(hydrophile/lipophile balance). Preferred surfactants of the
invention have a HLB of at least 10, preferably at least 15, and
more preferably at least 16. The invention can be used with
surfactants including, but not limited to: the polyoxyethylene
sorbitan esters surfactants (commonly referred to as the Tweens),
especially polysorbate 20 and polysorbate 80; copolymers of
ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide
(BO), sold under the DOWFAX.TM. tradename, such as linear EO/PO
block copolymers; octoxynols, which can vary in the number of
repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9
(Triton X-100, or t-octylphenoxypolyethoxyethanol) being of
particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL
CA-630/NP-40); phospholipids such as phosphatidylcholine
(lecithin); nonylphenol ethoxylates, such as the Tergitol.TM. NP
series; polyoxyethylene fatty ethers derived from lauryl, cetyl,
stearyl and oleyl alcohols (known as Brij surfactants), such as
triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters
(commonly known as the SPANs), such as sorbitan trioleate (Span 85)
and sorbitan monolaurate. Non-ionic surfactants are preferred.
Preferred surfactants for including in the emulsion are polysorbate
80 (polyoxyethylene sorbitan monooleate; Tween 80), Span 85
(sorbitan trioleate), lecithin and Triton X-100.
[0122] Mixtures of surfactants can be used e.g. Tween 80/Span 85
mixtures. A combination of a polyoxyethylene sorbitan ester such as
polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol
such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also
suitable. Another useful combination comprises laureth 9 plus a
polyoxyethylene sorbitan ester and/or an octoxynol.
[0123] Preferred amounts of surfactants (% by weight) are:
polyoxyethylene sorbitan esters (such as polysorbate 80) 0.01 to
1%, in particular about 0.1%; octyl- or nonylphenoxy
polyoxyethanols (such as Triton X-100, or other detergents in the
Triton series) 0.001 to 0.1%, in particular 0.005 to 0.02%;
polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably
0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
[0124] Specific oil-in-water emulsion adjuvants useful with the
invention include, but are not limited to: [0125] A submicron
emulsion of squalene, polysorbate 80, and sorbitan trioleate. The
composition of the emulsion by volume can be about 5% squalene,
about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate. In
weight terms, these ratios become 4.3% squalene, 0.5% polysorbate
80 and 0.48% sorbitan trioleate. This adjuvant is known as `MF59`
[108-110], as described in more detail in Chapter 10 of ref. 100
and chapter 12 of ref. 111. The MF59 emulsion advantageously
includes citrate ions e.g. 10 mM sodium citrate buffer. [0126] An
emulsion of squalene, a tocopherol, and polysorbate 80. The
emulsion may include phosphate buffered saline. It may also include
sorbitan trioleate (e.g. at 1%) and/or lecithin. These emulsions
may have from 2 to 10% squalene, from 2 to 10% tocopherol and from
0.3 to 3% polysorbate 80, and the weight ratio of
squalene:tocopherol is preferably .ltoreq.1 (e.g. 0.90) as this
provides a more stable emulsion. Squalene and polysorbate 80 may be
present volume ratio of about 5:2, or at a weight ratio of about
11:5. Thus the three components (squalene, tocopherol, polysorbate
80) may be present at a weight ratio of 1068:1186:485 or around
55:61:25. One such emulsion (`AS03`) can be made by dissolving
polysorbate 80 in PBS to give a 2% solution, then mixing 90 ml of
this solution with a mixture of (5 g of DL-.alpha.-tocopherol and 5
ml squalene), then microfluidising the mixture. The resulting
emulsion may have submicron oil droplets e.g. with an average
diameter of between 100 and 250 nm, preferably about 180 nm. The
emulsion may also include a 3-de-O-acylated monophosphoryl lipid A
(3d-MPL). Another useful emulsion of this type may comprise, per
human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4 mg
polysorbate 80 [112] e.g. in the ratios discussed above. [0127] An
emulsion of squalene, a tocopherol, and a Triton detergent (e.g.
Triton X-100). The emulsion may also include a 3d-MPL (see below).
The emulsion may contain a phosphate buffer. [0128] An emulsion
comprising a polysorbate (e.g. polysorbate 80), a Triton detergent
(e.g. Triton X-100) and a tocopherol (e.g. an .alpha.-tocopherol
succinate). The emulsion may include these three components at a
mass ratio of about 75:11:10 (e.g. 750 .mu.g/ml polysorbate 80, 110
.mu.g/ml Triton X-100 and 100 .mu.g/ml .alpha.-tocopherol
succinate), and these concentrations should include any
contribution of these components from antigens. The emulsion may
also include squalene. The emulsion may also include a 3d-MPL (see
below). The aqueous phase may contain a phosphate buffer. [0129] An
emulsion of squalane, polysorbate 80 and poloxamer 401
("Pluronic.TM. L121"). The emulsion can be formulated in phosphate
buffered saline, pH 7.4. This emulsion is a useful delivery vehicle
for muramyl dipeptides, and has been used with threonyl-MDP in the
"SAF-1" adjuvant [113] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic
L121 and 0.2% polysorbate 80). It can also be used without the
Thr-MDP, as in the "AF" adjuvant [114] (5% squalane, 1.25% Pluronic
L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
[0130] An emulsion comprising squalene, an aqueous solvent, a
polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g.
polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic
surfactant (e.g. a sorbitan ester or mannide ester, such as
sorbitan monoleate or `Span 80`). The emulsion is preferably
thermoreversible and/or has at least 90% of the oil droplets (by
volume) with a size less than 200 nm [115]. The emulsion may also
include one or more of: alditol; a cryoprotective agent (e.g. a
sugar, such as dodecylmaltoside and/or sucrose); and/or an
alkylpolyglycoside. It may also include a TLR4 agonist, such as one
whose chemical structure does not include a sugar ring [116]. Such
emulsions may be lyophilized. [0131] An emulsion having from
0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a
non-ionic surfactant. As described in reference 117, preferred
phospholipid components are phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidylglycerol, phosphatidic acid, sphingomyelin and
cardiolipin. Submicron droplet sizes are advantageous. [0132] A
submicron oil-in-water emulsion of a non-metabolisable oil (such as
light mineral oil) and at least one surfactant (such as lecithin,
polysorbate 80 or Span 80). Additives may be included, such as
QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as
GPI-0100, described in reference 118, produced by addition of
aliphatic amine to desacylsaponin via the carboxyl group of
glucuronic acid), dimethyidioctadecylammonium bromide and/or
N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine. [0133] An
emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g.
a cholesterol) are associated as helical micelles [119]. [0134] An
emulsion comprising a mineral oil, a non-ionic lipophilic
ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant
(e.g. an ethoxylated fatty alcohol and/or
polyoxyethylene-polyoxypropylene block copolymer) [120]. [0135] An
emulsion comprising a mineral oil, a non-ionic hydrophilic
ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant
(e.g. an ethoxylated fatty alcohol and/or
polyoxyethylene-polyoxypropylene block copolymer) [120].
[0136] Preferred oil-in-water emulsions used with the invention
comprise squalene and/or polysorbate 80.
[0137] The emulsions may be mixed with antigens during manufacture,
or they may be mixed extemporaneously at the time of delivery. Thus
the adjuvant and antigen may be kept separately in a packaged or
distributed vaccine, ready for final formulation at the time of
use. The antigen will generally be in an aqueous form, such that
the vaccine is finally prepared by mixing two liquids. The volume
ratio of the two liquids for mixing can vary (e.g. between 5:1 and
1:5) but is generally about 1:1. If emulsion and antigen are stored
separately in a multidose kit (from which multiple unit doses can
be taken) then the product may be presented as a vial containing
emulsion and a vial containing aqueous antigen, for mixing to give
adjuvanted liquid vaccine.
[0138] When used in formulating a vaccine, MF59 is preferably mixed
with antigens in phosphate-buffered saline to preserve the
long-term stability of MF59 formulations and to guarantee
physiological pH and osmolarity values in the final vaccine. This
mixing can be at a 1:1 volume ratio. The PBS can have pH 7.2.
[0139] Where a composition includes a tocopherol, any of the a, y,
8, c or tocopherols can be used, but .alpha.-tocopherols are
preferred. The tocopherol can take several forms e.g. different
salts and/or isomers. Salts include organic salts, such as
succinate, acetate, nicotinate, etc. D-.alpha.-tocopherol and
DL-.alpha.-tocopherol can both be used. Tocopherols are
advantageously included in vaccines for use in elderly patients
(e.g. aged 60 years or older) because vitamin E has been reported
to have a positive effect on the immune response in this patient
group. They also have antioxidant properties that may help to
stabilize the emulsions [121]. A preferred .alpha.-tocopherol is
DL-.alpha.-tocopherol, and the preferred salt of this tocopherol is
the succinate. The succinate salt has been found to cooperate with
TNF-related ligands in vivo.
[0140] Immunogenic Compositions
[0141] Compositions of the invention may comprise: (a) an antigenic
component; and (b) a non-antigenic component. The antigenic
component can comprise or consist of the antigens discussed above.
The non-antigenic component can include carriers, adjuvants,
excipients, buffers, etc. These non-antigenic components may have
various sources. For example, they may be present in one of the
antigen or adjuvant materials that is used during manufacture or
may be added separately from those components.
[0142] Preferred compositions of the invention include one or more
pharmaceutical carrier(s) and/or excipient(s).
[0143] To control tonicity, it is preferred to include a
physiological salt, such as a sodium salt. Sodium chloride (NaCl)
is preferred, which may be present at between 1 and 20 mg/ml.
[0144] Compositions will generally have an osmolality of between
200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg,
and will more preferably fall within the range of 280-320 mOsm/kg.
Osmolality has previously been reported not to have an impact on
pain caused by vaccination [122], but keeping osmolality in this
range is nevertheless preferred.
[0145] Compositions of the invention may include one or more
buffers. Typical buffers include: a phosphate buffer; a Tris
buffer; a borate buffer; a succinate buffer; a histidine buffer; or
a citrate buffer. Buffers will typically be included in the 5-20 mM
range.
[0146] A composition of the invention can be substantially free
from surfactants (prior to mixing with any emulsion adjuvant). In
particular, the composition of the invention can be substantially
free from polysorbate 80 e.g. it contains less than 0.1 .mu.g/ml of
polysorbate 80, and preferably contains no detectable polysorbate
80. Where a composition includes HBsAg, however, it will usually
include polysorbate 20 e.g. if it was used during yeast disruption
[29].
[0147] The pH of a composition of the invention will generally be
between 6.0 and 7.5. A manufacturing process may therefore include
a step of adjusting the pH of a composition prior to packaging.
[0148] Aqueous compositions administered to a patient can have a pH
of between 5.0 and 7.5, and more typically between 5.0 and 6.0 for
optimum stability; where a diphtheria toxoid and/or tetanus toxoid
is present, the pH is ideally between 6.0 and 7.0.
[0149] Compositions of the invention are preferably sterile.
[0150] Compositions of the invention are preferably non-pyrogenic
e.g. containing <1 EU (endotoxin unit, a standard measure; 1 EU
is equal to 0.2 ng FDA reference standard Endotoxin EC-2 `RSE`) per
dose, and preferably <0.1 EU per dose.
[0151] Compositions of the invention are preferably gluten
free.
[0152] Due to the adsorbed nature of antigens a vaccine product may
be a suspension with a cloudy appearance. This appearance means
that microbial contamination is not readily visible, and so the
vaccine preferably contains an antimicrobial agent. This is
particularly important when the vaccine is packaged in multidose
containers. Preferred antimicrobials for inclusion are
2-phenoxyethanol and thimerosal. It is preferred, however, not to
use mercurial preservative's (e.g. thimerosal) during the process
of the invention. Thus, between 1 and all of the components used in
the process may be substantially free from mercurial preservative.
However, the presence of trace amounts may be unavoidable if a
component was treated with such a preservative before being used in
the invention. For safety, however, it is preferred that the final
composition contains less than about 25 ng/ml mercury. More
preferably, the final vaccine product contains no detectable
thimerosal. This will generally be achieved by removing the
mercurial preservative from an antigen preparation prior to its
addition in the process of the invention or by avoiding the use of
thimerosal during the preparation of the components used to make
the composition. Mercury-free compositions are preferred.
[0153] Compositions of the invention will generally be in aqueous
form.
[0154] During manufacture, dilution of components to give desired
final concentrations will usually be performed with WFI (water for
injection).
[0155] The invention can provide bulk material which is suitable
for packaging into individual doses, which can then be distributed
for administration to patients. Concentrations discussed above are
typically concentrations in final packaged dose, and so
concentrations in bulk vaccine may be higher (e.g. to be reduced to
final concentrations by dilution).
[0156] Compositions of the invention are preferably administered to
patients in 0.5 ml unit doses. References to 0.5 ml doses will be
understood to include normal variance e.g. 0.5 ml.+-.0.05 ml. For
multidose situations, multiple dose amounts will be extracted and
packaged together in a single container e.g. 5 ml for a 10-dose
multidose container (or 5.5 ml with 10% overfill).
[0157] Residual material from individual antigenic components may
also be present in trace amounts in the final vaccine produced by
the process of the invention. For example, if formaldehyde is used
to prepare the toxoids of diphtheria, tetanus and pertussis then
the final vaccine product may retain trace amounts of formaldehyde
(e.g. less than 10 .mu.g/ml, preferably <5 .mu.g/ml). Media or
stabilizers may have been used during poliovirus preparation (e.g.
Medium 199), and these may carry through to the final vaccine.
Similarly, free amino acids (e.g. alanine, arginine, aspartate,
cysteine and/or cystine, glutamate, glutamine, glycine, histidine,
proline and/or hydroxyproline, isoleucine, leucine, lysine,
methionine, phenylalanine, serine, threonine, tryptophan, tyrosine
and/or valine), vitamins (e.g. choline, ascorbate, etc.), disodium
phosphate, monopotassium phosphate, calcium, glucose, adenine
sulfate, phenol red, sodium acetate, potassium chloride, etc. may
be retained in the final vaccine at .ltoreq.100 .mu.g/ml,
preferably <10 .mu.g/ml, each. Other components from antigen
preparations, such as neomycin (e.g. neomycin sulfate, particularly
from a poliovirus component), polymyxin B (e.g. polymyxin B
sulfate, particularly from a poliovirus component), etc. may also
be present at sub-nanogram amounts per dose. A further possible
component of the final vaccine which originates in the antigen
preparations arises from less-than-total purification of antigens.
Small amounts of B. pertussis, C. diphtheriae, C. tetani and S.
cerevisiae proteins and/or genomic DNA may therefore be present. To
minimize the amounts of these residual components, antigen
preparations are preferably treated to remove them prior to the
antigens being used with the invention.
[0158] Where a poliovirus component is used, it will generally have
been grown on Vero cells. The final vaccine preferably contains
less than 10 ng/ml, preferably .ltoreq.1 .mu.g/ml e.g. .ltoreq.500
pg/ml or .ltoreq.50 pg/ml of Vero cell DNA e.g. less than 10 ng/ml
of Vero cell DNA that is .gtoreq.50 base pairs long.
[0159] Compositions of the invention are presented for use in
containers. Suitable containers include vials and disposable
syringes (preferably sterile ones). Processes of the invention may
comprise a step of packaging the vaccine into containers for use.
Suitable containers include vials and disposable syringes
(preferably sterile ones).
[0160] Where a composition of the invention is presented in a vial,
this is preferably made of a glass or plastic material. The vial is
preferably sterilized before the composition is added to it. To
avoid problems with latex-sensitive patients, vials may be sealed
with a latex-free stopper. The vial may include a single dose of
vaccine, or it may include more than one dose (a `multidose` vial)
e.g. 10 doses. When using a multidose vial, each dose should be
withdrawn with a sterile needle and syringe under strict aseptic
conditions, taking care to avoid contaminating the vial contents.
Preferred vials are made of colorless glass.
[0161] A vial can have a cap (e.g. a Luer lock) adapted such that a
pre-filled syringe can be inserted into the cap, the contents of
the syringe can be expelled into the vial (e.g. to reconstitute
lyophilised material therein), and the contents of the vial can be
removed back into the syringe. After removal of the syringe from
the vial, a needle can then be attached and the composition can be
administered to a patient. The cap is preferably located inside a
seal or cover, such that the seal or cover has to be removed before
the cap can be accessed.
[0162] Where the composition is packaged into a syringe, the
syringe will not normally have a needle attached to it, although a
separate needle may be supplied with the syringe for assembly and
use. Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge
and 5/8-inch 25-gauge needles are typical. Syringes may be provided
with peel-off labels on which the lot number and expiration date of
the contents may be printed, to facilitate record keeping. The
plunger in the syringe preferably has a stopper to prevent the
plunger from being accidentally removed during aspiration. The
syringes may have a latex rubber cap and/or plunger. Disposable
syringes contain a single dose of vaccine.
[0163] The syringe will generally have a tip cap to seal the tip
prior to attachment of a needle, and the tip cap is preferably made
of butyl rubber. If the syringe and needle are packaged separately
then the needle is preferably fitted with a butyl rubber shield.
Grey butyl rubber is preferred. Preferred syringes are those
marketed under the trade name "Tip-Lok".TM..
[0164] Where a glass container (e.g. a syringe or a vial) is used,
then it is preferred to use a container made from a borosilicate
glass rather than from a soda lime glass.
[0165] After a composition is packaged into a container, the
container can then be enclosed within a box for distribution e.g.
inside a cardboard box, and the box will be labeled with details of
the vaccine e.g. its trade name, a list of the antigens in the
vaccine (e.g. `hepatitis B recombinant`, etc.), the presentation
container (e.g. `Disposable Prefilled Tip-Lok Syringes` or
`10.times.0.5 ml Single-Dose Vials`), its dose (e.g. `each
containing one 0.5 ml dose`), warnings (e.g. `For Adult Use Only`
or `For Pediatric Use Only`), an expiration date, an indication, a
patent number, etc. Each box might contain more than one packaged
vaccine e.g. five or ten packaged vaccines (particularly for
vials).
[0166] The vaccine may be packaged together (e.g. in the same box)
with a leaflet including details of the vaccine e.g. instructions
for administration, details of the antigens within the vaccine,
etc. The instructions may also contain warnings e.g. to keep a
solution of adrenaline readily available in case of anaphylactic
reaction following vaccination, etc.
[0167] The packaged vaccine is preferably stored at between
2.degree. C. and 8.degree. C. It should not be frozen.
[0168] Vaccines can be provided in full-liquid form (i.e. where all
antigenic components are in aqueous solution or suspension) after
manufacture, or they can be prepared in a form where the vaccine
can be prepared extemporaneously at the time/point of use by mixing
together two components. Such two-component embodiments include
liquid/liquid mixing and liquid/solid mixing e.g. by mixing aqueous
material with lyophilised material. For instance, in one embodiment
a vaccine can be made by mixing: (a) a first component comprising
aqueous antigens and/or adjuvant; and (b) a second component
comprising lyophilized antigens. In another embodiment a vaccine
can be made by mixing: (a) a first component comprising aqueous
antigens and/or adjuvant; and (b) a second component comprising
aqueous antigens. In another embodiment a vaccine can be made by
mixing: (a) a first component comprising aqueous antigens; and (b)
a second component comprising aqueous adjuvant. The two components
are preferably in separate containers (e.g. vials and/or syringes),
and the invention provides a kit comprising components (a) and
(b).
[0169] Another useful liquid/lyophilised format comprises (a) an
oil-in-water emulsion adjuvant and (b) a lyophilised component
including one or more antigens. A vaccine composition suitable for
patient administration is obtained by mixing components (a) and
(b). In some embodiments component (a) is antigen-free, such that
all antigenic components in the final vaccine are derived from
component (b); in other embodiments component (a) includes one or
more antigen(s), such that the antigenic components in the final
vaccine are derived from both components (a) and (b).
[0170] Another useful liquid/lyophilised format comprises (a) an
aqueous complex of an aluminium salt and a TLR agonist and (b) a
lyophilised component including one or more antigens. A vaccine
composition suitable for patient administration is obtained by
mixing components (a) and (b). In some embodiments component (a) is
antigen-free, such that all antigenic components in the final
vaccine are derived from component (b); in other embodiments
component (a) includes one or more antigen(s), such that the
antigenic components in the final vaccine are derived from both
components (a) and (b).
[0171] Thus the invention provides a kit for preparing a
combination vaccine, comprising components (a) and (b) as noted
above. The kit components are typically vials or syringes, and a
single kit may contain both a vial and a syringe. The invention
also provides a process for preparing such a kit, comprising the
following steps: (i) preparing an aqueous component vaccine as
described above; (ii) packaging said aqueous combination vaccine in
a first container e.g a syringe; (iii) preparing an
antigen-containing component in lyophilised form; (iv) packaging
said lyophilised antigen in a second container e.g. a vial; and (v)
packaging the first container and second container together in a
kit. The kit can then be distributed to physicians.
[0172] A liquid/lyophilised format is particularly useful for
vaccines that include a conjugate component, particularly Hib
and/or meningococcal and/or pneumococcal conjugates, as these may
be more stable in lyophilized form. Thus conjugates may be
lyophilised prior to their use with the invention.
[0173] Where a component is lyophilised it generally includes
non-active components which were added prior to freeze-drying e.g.
as stabilizers. Preferred stabilizers for inclusion are lactose,
sucrose and mannitol, as well as mixtures thereof e.g.
lactose/sucrose mixtures, sucrose/mannitol mixtures, etc. A final
vaccine obtained by aqueous reconstitution of the lyophilised
material may thus contain lactose and/or sucrose. It is preferred
to use amorphous excipients and/or amorphous buffers when preparing
lyophilised vaccines [123].
[0174] Preferred compositions of the invention include (1)
diphtheria, tetanus and pertussis toxoids, inactivated poliovirus
for Types 1, 2 & 3, plus (2) hepatitis B virus surface antigen
and/or a Hib conjugate. These compositions may consist of the
antigens specified, or may further include antigens from additional
pathogens (e.g. meningococcus). Thus the compositions can be used
as vaccines themselves, or as components of further vaccines.
[0175] Where a composition includes both diphtheria and tetanus
toxoids these may be present at various ratios. There is preferably
an excess of diphtheria toxoid (measured in Lf units) e.g. between
2-4.times. more diphtheria toxoid than tetanus toxoid, such as
2.5.times. or 3.times. more.
[0176] Methods of Treatment, and Administration of the Vaccine
[0177] Compositions of the invention are suitable for
administration to human patients, and the invention provides a
method of raising an immune response in a patient, comprising the
step of administering a composition of the invention to the
patient.
[0178] The invention also provides a composition of the invention
for use in medicine.
[0179] The invention also provides the use of (i) at least a
diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid and
(ii) an aluminium salt adjuvant, in the manufacture of a
combination vaccine which includes less than 0.2 mg Al.sup.+++ per
unit dose.
[0180] The invention also provides the use of (i) at least a
diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid and
(ii) an aluminium salt adjuvant, in the manufacture of a
combination vaccine which includes a low dose of each of a
diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid.
[0181] The invention also provides the use of (i) at least a
diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid and
(ii) an aluminium salt adjuvant, in the manufacture of a
combination vaccine which includes a low dose of each of a
diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid and has
less than 0.2 mg Al.sup.+++ per unit dose.
[0182] The invention also provides the use of (i) a diphtheria
toxoid, a tetanus toxoid, a pertussis toxoid, and a Hib conjugate
(ii) a hepatitis B virus surface antigen and/or an inactivated
poliovirus antigen, and (iii) an oil-in-water emulsion adjuvant, in
the manufacture of a combination vaccine.
[0183] Immunogenic compositions of the invention are preferably
vaccines, for use in the prevention of at least diphtheria,
tetanus, whooping cough. Depending on their antigen content the
vaccines may also protect against bacterial meningitis, polio,
hepatitis, etc.
[0184] In order to have full efficacy, a typical primary
immunization schedule (particularly for a child) may involve
administering more than one dose. For example, doses may be at: 0
& 6 months (time 0 being the first dose); at 0, 1, 2 & 6
months; at day 0, day 21 and then a third dose between 6 & 12
months; at 2, 4 & 6 months; at 3, 4 & 5 months; at 6, 10
& 14 weeks; at 2, 3 & 4 months; or at 0, 1, 2, 6 & 12
months.
[0185] Compositions can also be used as booster doses e.g. for
children, in the second year of life.
[0186] Compositions of the invention can be administered by
intramuscular injection e.g. into the arm or leg.
[0187] Infant Immunisation Schedule with Fewer Doses
[0188] As mentioned above, a further aspect of the invention is an
immunisation schedule for an infant (i.e. a child between birth and
1 year of age) in which only one or two DTP-containing compositions
are administered. Thus, in some embodiments, the invention delivers
fewer doses compared to the current normal 3-dose schedule, but
without loss of immunoprotective effect.
[0189] According to this aspect, therefore, the invention provides:
[0190] a method for immunising an infant against at least
diphtheria, tetanus and pertussis (whooping cough), comprising
administering to the infant no more than two doses of a combination
vaccine comprising a diphtheria toxoid, a tetanus toxoid, and a
pertussis toxoid. [0191] a method for immunising an infant against
at least diphtheria, tetanus and pertussis (whooping cough),
comprising administering to the infant no more than two doses of a
combination vaccine comprising a diphtheria toxoid, a tetanus
toxoid, a pertussis toxoid, and an aluminium salt adjuvant, wherein
each dose of the vaccine contains less than 0.2 mg Al.sup.+++.
[0192] a method for immunising an infant against at least
diphtheria, tetanus and pertussis (whooping cough), comprising
administering to the infant no more than two doses of a combination
vaccine comprising an aluminium salt adjuvant and a low dose of
each of a diphtheria toxoid, a tetanus toxoid, and a pertussis
toxoid. [0193] a method for immunising an infant against at least
diphtheria, tetanus and pertussis (whooping cough), comprising
administering to the infant no more than two doses of a combination
vaccine comprising (i) a low dose of each of a diphtheria toxoid, a
tetanus toxoid, and a pertussis toxoid, and (ii) an aluminium salt
adjuvant; wherein each dose of the vaccine contains less than 0.2
mg Al.sup.+++. [0194] a method for immunising an infant against at
least diphtheria, tetanus and pertussis (whooping cough),
comprising administering to the infant no more than two doses of a
aluminium-free combination vaccine comprising a diphtheria toxoid,
a tetanus toxoid, a pertussis toxoid, and an oil-in-water emulsion
adjuvant. The vaccine may have a low dose of each of a diphtheria
toxoid, a tetanus toxoid, and a pertussis toxoid. [0195] use of at
least a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid
in the manufacture of a combination vaccine for immunising an
infant against at least diphtheria, tetanus and pertussis, wherein
the vaccine is prepared for administration to the infant by no more
than two doses. The vaccine may: (i) include an aluminium salt
adjuvant, in which case it may include less than 0.2 mg Al.sup.+++
per unit dose; and/or (ii) have a low dose of each of a diphtheria
toxoid, a tetanus toxoid, and a pertussis toxoid. [0196] use of at
least a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid
in the manufacture of an aluminium-free combination vaccine for
immunising an infant against at least diphtheria, tetanus and
pertussis, wherein the vaccine is prepared for administration to
the infant by no more than two doses. The vaccine may comprise,
either during manufacture or at the point of use, an oil-in-water
emulsion adjuvant. The vaccine may have a low dose of each of a
diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid. [0197]
a combination vaccine comprising at least a diphtheria toxoid, a
tetanus toxoid, and a pertussis toxoid, for use in a method for
immunising an infant against at least diphtheria, tetanus and
pertussis (whooping cough) by administering to the infant no more
than two doses of the combination vaccine. The vaccine may: (i)
include an aluminium salt adjuvant, in which case it may include
less than 0.2 mg Al.sup.+++ per unit dose; and/or (ii) have a low
dose of each of a diphtheria toxoid, a tetanus toxoid, and a
pertussis toxoid. [0198] an aluminium-free combination vaccine
comprising at least an oil-in-water emulsion adjuvant, a diphtheria
toxoid, a tetanus toxoid, and a pertussis toxoid, for use in a
method for immunising an infant against at least diphtheria,
tetanus and pertussis (whooping cough) by administering to the
infant no more than two doses of the combination vaccine. The
vaccine may have a low dose of each of a diphtheria toxoid, a
tetanus toxoid, and a pertussis toxoid.
[0199] According to this aspect, where the vaccine includes an
aluminium salt adjuvant then, as disclosed above, the vaccine can
also include a TLR agonist which may be adsorbed to that aluminium
salt.
[0200] According to this aspect, the combination vaccine includes a
pertussis toxoid. This may be incorporated into the vaccine as a
protein within a cellular pertussis antigen, but it is preferred to
use an acellular pertussis antigen, as discussed in more detail
above.
[0201] According to this aspect, no more than two doses of the
vaccine are given to the infant i.e. the infant receives a single
dose or two doses of the vaccine, but does not receive three (or
more) doses. The infant may, though, receive a third (and maybe
further) dose later in their life i.e. after their first birthday
or after their second birthday.
[0202] The one or two dose(s) is/are preferably given to the infant
(i) between 1 and 5 months of age (ii) between 2 and 4 months of
age (iii) between 3 and 5 months of age (iv) between 6 and 16 weeks
of age or (v) between 0 and 3 months of age. For instance, two
doses may be given at (i) 1 & 2 months of age (ii) 2 & 4
months of age (iii) 3 & 4 months of age (iv) 2 & 3 months
of age (v) 0 and 1 months of age, etc.
[0203] General
[0204] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0205] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0206] The term "about" in relation to a numerical value x means,
for example, x.+-.10%.
[0207] Unless specifically stated, a process comprising a step of
mixing two or more components does not require any specific order
of mixing. Thus components can be mixed in any order. Where there
are three components then two components can be combined with each
other, and then the combination may be combined with the third
component, etc.
[0208] Where an antigen is described as being "adsorbed" to an
adjuvant, it is preferred that at least 50% (by weight) of that
antigen is adsorbed e.g. 50%, 60%, 70%, 80%, 90%, 95%, 98% or more.
It is preferred that diphtheria toxoid and tetanus toxoid are both
totally adsorbed i.e. none is detectable in supernatant. Total
adsorption of HBsAg can be used.
[0209] Amounts of conjugates are generally given in terms of mass
of saccharide (i.e. the dose of the conjugate (carrier+saccharide)
as a whole is higher than the stated dose) in order to avoid
variation due to choice of carrier.
[0210] Where a composition includes an aluminium salt adjuvant then
preferably it does not also include an oil-in-water emulsion
adjuvant. Conversely, where a composition includes an oil-in-water
emulsion adjuvant then preferably it does not also include an
aluminium salt adjuvant.
[0211] Phosphorous-containing groups employed with the invention
may exist in a number of protonated and deprotonated forms
depending on the pH of the surrounding environment, for example the
pH of the solvent in which they are dissolved. Therefore, although
a particular form may be illustrated herein, it is intended, unless
otherwise mentioned, for these illustrations to merely be
representative and not limiting to a specific protonated or
deprotonated form. For example, in the case of a phosphate group,
this has been illustrated as --OP(O)(OH).sub.2 but the definition
includes the protonated forms --[OP(O)(OH.sub.2)(OH)].sup.+ and
--[OP(O)(OH.sub.2).sub.2].sup.2+ that may exist in acidic
conditions and the deprotonated forms --[OP(O)(OH)(O)].sup.- and
[OP(O)(O).sub.2].sup.2- that may exist in basic conditions. The
invention encompasses all such forms.
[0212] TLR agonists can exist as pharmaceutically acceptable salts.
Thus, the compounds may be used in the form of their
pharmaceutically acceptable salts i.e. physiologically or
toxicologically tolerable salt (which includes, when appropriate,
pharmaceutically acceptable base addition salts and
pharmaceutically acceptable acid addition salts).
[0213] In the case of TLR agonists shown herein which may exist in
tautomeric forms (i.e. in keto or enol forms), the compound can be
used in all such tautomeric forms.
[0214] Where a compound is administered to the body as part of a
composition then that compound may alternatively be replaced by a
suitable prodrug.
[0215] Where animal (and particularly bovine) materials are used in
the culture of cells, they should be obtained from sources that are
free from transmissible spongiform encephalopathies (TSEs), and in
particular free from bovine spongiform encephalopathy (BSE).
BRIEF DESCRIPTION OF THE DRAWINGS
[0216] There are no drawings.
MODES FOR CARRYING OUT THE INVENTION
[0217] Adjuvant Adsorption to Antigens
[0218] 3-valent (DTaP) or 6-valent (DTaP-HBsAg--IPV-Hib) vaccines
were adjuvanted with aluminium hydroxide alone, aluminium hydroxide
with pre-adsorbed `compound T`, poly(lactide-co-glycolide)
microparticles (`PLG`), and MF59 oil-in-water emulsion. Aluminium
hydroxide and aluminium hydroxide with pre-adsorbed `compound T`
were prepared in histidine buffer pH 6.5. At pH 6.5, aluminium
hydroxide has a positive net charge, while most proteins have a
negative net charge. The pH value was chosen to provide good
adsorption of most of the tested antigens. All formulations
adjuvanted with aluminium hydroxide or aluminium hydroxide with
pre-adsorbed `compound T` showed optimal pH (6.5-6.8.+-.0.1) and
osmolarity values (0.300.+-.50 mO). Osmolarity was adjusted with
NaCl. Antigens for the MF59-adjuvanted formulations were prepared
in PBS. The resulting preparations had pH values between 6.2 and
7.3 and osmolarity values around 0.300.+-.50 mO. Formulations
containing PLG microparticles were prepared in water. PLG
formulations showed suboptimal osmolarity values. The pH of the PLG
formulations ranged from 5.8 to 6.5+0.1. The PLG microparticles
were prepared with dioctylsulfosuccinate (DSS) which confers a
negative net charge to the microparticles. Thus interaction of the
microparticles with the antigen is mediated by positive charges on
the antigen surface.
[0219] For aluminium hydroxide alone, aluminium hydroxide with
pre-adsorbed `compound T`, and PLG, adsorption was detected by
separating the adjuvant-antigen complexes from unadsorbed antigens
by centrifugation. 0.4% DOC was added to the supernatant containing
the unadsorbed antigens.
[0220] Antigens were precipitated by the addition of 60% TCA and
collected by centrifugation. The pellet containing the
TCA-precipitated antigens was resuspended in loading buffer and
loaded onto an SDS-PAGE gel. The pellet containing the
adjuvant-antigen complexes was resuspended in desorption buffer
(4.times. concentration: 0.5 M Na.sub.2HPO.sub.4 pH, 8 g SDS, 25 g
glycerol, 6.16 g DTT and bromophenol blue), the aluminium hydroxide
was removed by centrifugation and the supernatant applied to an
SDS-PAGE gel. The MF59 oil-in-water emulsion containing antigens
were separated by centrifugation in an oily phase and an aqueous
phase. Both the aqueous phase containing unabsorbed antigens and
the oily phase presumably containing MF59-associated antigens were
mixed with loading buffer and applied to an SDS-PAGE gel. After
electrophoretic separation of the samples, the gels were either
analysed by Coomassie Blue staining or by Western blotting.
[0221] Using aluminium hydroxide alone at a concentration of 2
mg/ml, the adsorption profiles for DT, TT, PT, FHA and 69K detected
by Coomassie Blue staining were complete both for the 3-valent
formulation and the 6-valent formulation. No bands were detected in
the DOC-TCA-treated supernatants. Western Blot analysis confirmed
complete aluminium hydroxide adsorption for DT, TT, PT, FHA and 69K
for both the 3-valent formulation and the 6-valent formulation.
Likewise, the other five antigens--IPV1, IPV2, IPV3, HBsAg and
Hib-CRM--did not show any detectable bands in the DOC-TCA-treated
supernatants of aluminium hydroxide-adsorbed formulations. Thus all
ten antigens present in the 6-valent formulation completely
adsorbed to aluminium hydroxide.
[0222] For aluminium hydroxide with pre-adsorbed `compound T`,
antigen adsorption differed between the 3-valent formulation and
the 6-valent formulation. Four different `compound T`
concentrations were tested (0.1, 0.025, 0.01, 0.005 mg/ml). The
aluminium hydroxide concentration was kept constant at 2 mg/ml. At
0.1 mg/ml `compound T`, all antigens in the 3-valent formulation
were completely adsorbed. In contrast, 69K and PT in the 6-valent
formulation were not completely adsorbed as determined by Coomassie
Blue staining. At 0.01 mg/ml `compound T`, Western blot analysis
confirmed adsorption of all ten antigens in the 6-valent
formulation. Only a small amount of TT was still detectable in the
supernatant using Western blot. The fact that TT could be detected
in the supernatant by Western blot but not by SDS-PAGE is likely
due to the greater sensitivity of the former method. Thus, at
higher concentrations, `compound T` appears to compete with the
antigens for binding to the adjuvant. This could explain why the
effect only becomes apparent in the presence of a greater number of
antigens, i.e., when less aluminium hydroxide per antigen is
available.
[0223] Using PLG microparticles, DT, TT, IPV1, IPV2, IPV3, FHA and
CRM of the Hib-CRM conjugate were mostly presented on the
supernatants with only very small amounts of DT, IPV1, IPV2 and FHA
being detected by Western blot in the pellet containing the
antigen-adjuvant complexes. 69K and PT seemed to be presented in
similar amounts in supernatant and pellet. HBsAg could neither be
detected in the supernatant nor in the pellet of the PLG
formulations. In comparison to preparations containing aluminium
hydroxide or aluminium hydroxide with pre-adsorbed `compound T`,
PLG absorbed significantly less antigen. Moreover, the antigen
adsorption profiles obtained using PLG showed an opposite trend to
those seen in the presence of the other two adjuvants probably
reflecting the negative net charge of PLG versus the positive net
charge of aluminium hydroxide or aluminium hydroxide with
pre-adsorbed `compound T`.
[0224] MF59 is a delivery system generally considered unable to
physically interact with the antigens as shown by the lack of an
antigen deposition at the injection site and independent clearance
of MF59 and the antigens (see references 124 and 125). 1:1, 1:3 and
1:10 ratios (v:v of MF59 to complete antigen formulation) were
tested. For all three tested ratios, SDS-PAGE and Western blot
analysis showed that all ten tested antigens were present in the
aqueous phase of MF59-adjuvanted formulations. Thus the antigen
profiles of MF59-adjuvanted formulations corresponded to the
profiles of unadjuvanted formulations. The results confirmed that
MF59 does not interact with any of the tested antigens.
[0225] Replacement or Reduction of Aluminium Salt Adjuvants
[0226] The INFANRIX HEXA product from GlaxoSmithKline contains
.gtoreq.30 IU diphtheria toxoid, .gtoreq.40 IU tetanus toxoid, an
acellular pertussis component (25/25/8 .mu.g of PT/FHA/pertactin),
10 .mu.g HBsAg, a trivalent IPV component (40/8/32 DU of types
1/2/3), and 10 .mu.g Hib conjugate. The vaccine is presented as a
5-valent aqueous vaccine which is used to reconstitute the Hib
conjugate from its lyophilised form, to give a 0.5 ml aqueous unit
dose for human infants which contains 0.95 mg aluminium hydroxide
and 1.45 mg aluminium phosphate.
[0227] To investigate alternative adjuvants (see above) a 6-valent
mixture was adjuvanted with aluminium hydroxide alone (2 mg/ml in
histidine buffer), with aluminium hydroxide with pre-adsorbed
`compound T` (see above; lmg/ml), with poly(lactide-co-glycolide)
microparticles (`PLG`, used at 40 mg/ml), or with the MF59
oil-in-water emulsion (mixed at equal volume with antigens in
phosphate-buffered saline). The same diluents were used in all
mouse experiments described below. Osmolarity of the formulations
was adjusted with NaCl where necessary. An adjuvant-free control
was also prepared. Antigen concentrations were as follows (per
ml):
TABLE-US-00003 DT TT PT FHA Pertactin 36.9 Lf 14.8 Lf 36.9 .mu.g
36.9 .mu.g 11.8 .mu.g IPV Type 1 IPV Type 2 IPV Type 3 HBsAg Hib
59.1 DU 11.8 DU 47.3 DU 14.8 .mu.g 14.8 .mu.g
[0228] The same adjuvants were also used with a 3-valent D-T-Pa
mixture (same concentrations).
[0229] Osmolarity and pH were measured (and, if necessary,
adjusted) after combining the components in order to ensure
physiological acceptability. For all 3-valent compositions the pH
was between 5.9 and 7.1 and osmolarity was between 290-320 mOsm/kg
(except one at >400 mOsm/kg). For all 6-valent compositions the
pH was between 5.5 and 6.8 and osmolarity was between 260-320
mOsm/kg (except one at >500 mOsm/kg). A buffer control had pH
7.3 and 276 mOsm/kg.
[0230] The integrity and immunogenicity of the combined antigens
were also tested. None of antigens showed an altered analytical
profile after being formulated as combinations i.e. the antigens
and adjuvants are physically compatible together.
[0231] With aluminium hydroxide alone all antigens adsorbed well to
the adjuvant. With aluminium hydroxide and compound `T` (i.e.
aluminium hydroxide which had been pre-mixed with `compound T` to
permit adsorption for formation of a stable adjuvant complex;
hereafter) all antigens adsorbed well, except that TT, pertactin
and PT were partially desorbed.
[0232] With the PLG adjuvant the diphtheria and tetanus toxoids
were unadsorbed but pertussis toxoid was adsorbed.
[0233] Mice (female Balb/c, 4 weeks old) were immunised
intramuscularly with 100 .mu.l of each composition (i.e. 1/5 human
dose volume) at days 0 and 28. Sera were collected 14 days after
each injection. After the second immunisation IgG antibody titers
were as follows:
TABLE-US-00004 Al No hydrox- Infanrix- adjuvant ide MF59 PLG Al-T 6
3-valent vaccines DT 750 21626 15693 9430 23395 -- TT 13120 17868
22458 15917 23131 -- Pertactin 639 7209 10258 3946 12857 -- PT 2501
8270 7212 3679 9938 -- FHA 3982 12057 14098 14139 23008 -- 6-valent
vaccine DT 1751 18914 13982 7658 23102 21581 TT 12729 16756 22229
13744 23267 15998 Pertactin 333 6299 9363 2912 5153 10809 PT 3069
3384 4823 3906 6484 6052 FHA 4558 7206 16201 15206 19383 11051 Hib
177 813 1266 654 2153 1269 HBsAg 1058 1598 2288 1053 4501 1113
[0234] Thus for all of these antigens the inclusion of an adjuvant
increased IgG antibody titers. The best titers were seen when using
Al-T. The next best were with MF59, which gave better results than
aluminium hydroxide alone. The titers obtained using Al-T were
better for all antigens than those seen with Infanrix Hexa, except
for pertactin.
[0235] Furthermore, the data show that the good results achieved
with the 3-valent vaccine are maintained even after IPV, Hib and
HBsAg are added.
[0236] IgG responses were also investigated by subclass. For most
of the antigens in the 6-valent vaccines the adjuvants had little
effect on IgG1 titers, but they did increase IgG2a and IgG2b
titers. The best IgG2a and IgG2b titers were obtained with Al-T,
and then with MF59.
[0237] The increased titers seen with Al-T compared with aluminium
hydroxide alone, or with the mixture of aluminium salts seen in
Infanrix Hexa, mean that the total amount of aluminium per dose can
be reduced while maintaining enhancement of immune responses.
[0238] Reduction of Antigen Doses
[0239] Experiments were designed to investigate whether the
improved adjuvants could be used to reduce the amount of antigen
per dose. 10-fold, 50-fold and 100-fold dilutions (relative to
human dosing i.e. to deliver 1 .mu.g, 0.2 .mu.g or 0.1 .mu.g HBsAg
to each mouse per 100 .mu.l dose) of the 6-valent antigen
combinations were made while adjuvant concentration was
maintained.
[0240] Osmolarity and pH were measured (and, if necessary,
adjusted) after dilution. For all 6-valent compositions the pH was
between 6.1 and 7.0 and osmolarity was between 275-320 mOsm/kg. A
buffer control had pH 7.3 and 285 mOsm/kg.
[0241] Mice were immunised in the same way as discussed above.
Total serum IgG titers after 2 immunisations were as follows:
TABLE-US-00005 No adjuvant Al hydroxide MF59 Al-T 1/10 1/50 1/100
1/10 1/50 1/100 1/10 1/50 1/100 1/10 1/50 1/100 DT 459 2043 137
18357 13106 7541 17431 6003 8736 21913 16807 13724 TT 7602 7929
1700 17595 9664 5531 22791 12062 13015 23570 12237 13183 Pertactin
827 2154 341 10880 8135 4181 17159 10591 7288 17098 10748 8952 PT
3612 5645 2129 5287 3266 1068 7200 3659 5493 9051 4203 2717 FHA
2305 4161 101 8997 4471 1442 19197 5179 4492 22151 8293 3252 Hib
171 352 109 1380 796 251 3147 573 2415 3056 1440 1815 HBsAg 525 412
129 1034 685 226 4885 1103 1983 5270 1526 950
[0242] Thus the presence of adjuvants allowed a dose reduction of
5-fold or 10-fold while maintaining IgG titers which are comparable
or higher to unadjuvanted antigens. MF59 and Al-T in particular are
useful for dose sparing of antigens in this manner.
[0243] Adjuvant Dosing
[0244] With the 100-fold antigen dilution the amount of adjuvant
was also reduced. The MF59 emulsion was mixed with antigens at a
1:1 volume ratio or at a 1:3 ratio (i.e. 1 ml of emulsion for every
3 ml of antigen, with 2 ml of buffer to maintain total volume) or
at a 1:10 ratio. The Al-T complex was prepared at 3 strengths
having 2 mg/ml aluminium hydroxide with either 5 .mu.g, 25 .mu.g or
100 .mu.g of `compound T` per dose. For comparison a 1:100 antigen
dose was tested in unadjuvanted form or with aluminium hydroxide
alone. A 1:100 dilution of Infanrix Hexa was also used for
comparison.
[0245] Osmolarity and pH were measured (and, if necessary,
adjusted) after mixing (except for Infanrix Hexa). For all 6-valent
compositions the pH was between 6.2 and 7.3 and osmolarity was
between 270-320 mOsm/kg. A buffer control had pH 7.3 and 280
mOsm/kg.
[0246] Mice were immunised as before. Total serum IgG titers after
2 immunisations were as follows:
TABLE-US-00006 No Infanrx Al MF59 (v:v) Al-T (.mu.g `T`) adjuvant
Hexa hydroxide 1:1 1:3 1:10 100 25 5 DT 584 6282 10849 7786 4094
8442 21571 20865 11788 TT 3426 5415 6857 11506 9197 11422 16041
15124 6236 Pertactin 48 3017 6053 8838 2970 2876 6158 6697 3815 PT
3351 1751 2699 4406 5072 6020 2476 2696 3079 FHA 262 7886 5626
14700 11340 10205 7369 8634 6120 Hib 126 109 310 518 517 550 936
792 390 HBsAg 88 240 369 2645 1784 1670 4062 2308 1154
[0247] Thus lower amounts of MF59 and Al-T still retain good
adjuvanticity and can induce higher IgG antibody titers than those
induced by unadjuvanted 6-valent antigen formulations. By reducing
the amount of adjuvant, while maintaining immunological efficacy,
the safety profile of a vaccine can be improved which is
particularly important in pediatric settings.
[0248] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
TABLE-US-00007 TABLE A antigen and Al.sup.+++ content of various
marketed vaccines (per unit dose) D T Pa.sup.(1) Hib.sup.(2)
IPV.sup.(3) HBsAg Vol Al.sup.+++ Pediacel 15 Lf 5 Lf 20/20/3 10
40/8/32 -- 0.5 ml 0.33 mg Pediarix 25 Lf 10 Lf 25/25/8 -- 40/8/32
10 .mu.g 0.5 ml .ltoreq.0.85 mg Pentacel 15 Lf 5 Lf 20/20/3 10
40/8/32 -- 0.5 ml 0.33 mg Tritan.sup.x HB .gtoreq.30 IU .gtoreq.60
IU --.sup.(4) -- -- 10 .mu.g 0.5 ml 0.63 mg Quinvaxem .gtoreq.30 IU
.gtoreq.60 IU --.sup.(4) 10 -- 10 .mu.g 0.5 ml 0.3 mg Hexavac 30 Lf
10 Lf 25/25/-- 12 40/8/32 5 .mu.g 0.5 ml 0.3 mg Boostrix 2.5 Lf 5
Lf 8/8/2.5 -- -- -- 0.5 ml .ltoreq.0.39 mg Adacel 5 Lf 2 Lf 2.5/5/3
-- -- -- 0.5 ml 0.33 mg Daptacel 15 Lf 5 Lf 10/5/3 -- -- -- 0.5 ml
0.33 mg Pentavac .gtoreq.30 IU .gtoreq.40 IU 25/25/-- 10 40/8/32 --
0.5 ml 0.30 mg SII QVac 20-30 Lf 5-25 Lf --.sup.(4) -- --
.gtoreq.10 .mu.g 0.5 ml .ltoreq.1.25 mg TripVacHB .gtoreq.30 IU
.gtoreq.60 IU --.sup.(4) -- -- 10 .mu.g 0.5 ml .ltoreq.1.25 mg
Notes: .sup.(1)Pa dose shows amounts of pertussis toxoid, then FHA,
then pertactin (.mu.g). Pediacel's, Daptacel's and Adacel's Pa
components also contain fimbriae types 2 and 3. .sup.(2)Hib dose
shows amount of PRP capsular saccharide (.mu.g). .sup.(3)IPV dose
shows amounts of type 1, then type 2, then type 3 (measured in DU).
.sup.(4)Tritanrix-HepB, Quinvaxem, Trip Vac HB and SII Q-Vac
include whole-cell pertussis antigens
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