Note: Descriptions are shown in the official language in which they were submitted.
CA 02462646 2011-02-22
ADJUVANTED MENINGOCOCCUS COMPOSITIONS
TECHNICAL FIELD
The invention relates to vaccines, more particularly those against Neisseria
nieningitidis.
BACKGROUND ART
Genome sequences for Neisseria meningitidis (meningococcus) serogroups A [1]
and B [2,3]
have been reported. The serogroup B sequence has been studied to identify
vaccine antigens [e.g.
refs. 4 to 9] and candidate antigens have been manipulated to improve
heterologous expression [refs.
10 to 12].
Antigens generally require the co-administration of adjuvants in order to
enhance their
immunogenicity in vaccines [13]. Freund's adjuvant has been used for serogroup
B meningococcus
[9], and the licensed vaccine MenjugateTM against serogroup C uses aluminium
hydroxide [14].
Enhancement of the bactericidal activity of Neisseria antigens has also been
reported by using
oligonucleotide adjuvants containing CpG motifs [15].
It is an object of the invention to provide further and improved adjuvants for
Neisserial
antigens.
DISCLOSURE OF THE INVENTION
It has been found that a combination of CpG oligonucleotides and polymer
microparticles is
an extremely effective adjuvant for Neisserial antigens, with the combination
giving much better
results than either of the individual components. The invention therefore
provides a composition
comprising: (a) a Neisserial antigen; (b) a CpG oligonucleotide; and (c) a
biodegradable polymer
microparticle.
The Neisserial antigen
The Neisserial antigen may be a protein antigen, nucleic acid encoding a
protein antigen, or a
saccharide antigen. The antigen preferably elicits a bactericidal or
protective immune response (e.g.
antibody response) in a recipient mammal.
The antigen may be derived from any species of Neisseria including
N.gonorrhoeae,
N.lactanxica and N.meningitidis. It is preferably a N.meningitidis antigen and
may be from any
serogroup. Where the antigen is from serogroup B, it is preferred to use a
protein antigen; where it is
from serogroup A, C, W135 or Y then it is preferred to use a saccharide
antigen. Where saccharide
antigens are used, these will typically be derived from the bacterial capsular
polysaccharide (e.g.
oligosaccharides, such as those obtained by hydrolysis), and they will
typically be conjugated to
carrier proteins (e.g. to CRM197).
1
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
Preferred protein antigens derived from serogroup B N.meningitidis are:
= a protein disclosed in any one of references 4, 5, 6, 7, 8 or 9 (in
particular the 446 even SEQ
IDs (i.e. 2, 4, 6, ... , 890, 892) disclosed in reference 4, the 45 even SEQ
IDs (i.e. 2, 4, 6, ... ,
88, 90) disclosed in reference 5 and the 1674 even SEQ IDs 2-3020, even SEQ
IDs 3040-
3114, and all SEQ IDs 3115-3241, disclosed in reference 6);
= a protein comprising an immunogenic fragment of one or more of the proteins
disclosed in
any one of references 4, 5, 6, 7, 8 or 9.
= a protein comprising a sequence having sequence identity (preferably greater
than 50% e.g.
60%, 70%, 80%, 90%, 95%, 99% or more) to one or more of the proteins disclosed
in any
one of references 4, 5, 6, 7, 8 or 9.
= a protein disclosed in any one of references 10, 11 or 12.
= a protein comprising a sequence having sequence identity (preferably greater
than 50% e.g.
60%, 70%, 80%, 90%, 95%, 99% or more) to one or more of the proteins disclosed
in any
one of references 10, 11 or 12.
A particularly preferred protein antigen from serogroup B N.meningitidis is
protein `287'.
This protein may be used in a wild-type form [e.g. GenBank accession
gi:7228690; alignments of
polymorphic forms of 287 are shown in figures 5 & 15 of ref. 8] but
derivatives of the wild-type
protein may be used. For instance, proteins having 50% or more sequence
identity (e.g. 60%, 70%,
80%, 90%, 95%, 99% or more) to gi:7228690 may be used. Proteins comprising
truncation or
deletion variants of the protein may be used, such as the N-terminal truncated
forms disclosed in
references 10 to 12 ('AG287' in particular, in which the N-terminus of the
protein up to and
including the six repeated glycine residues is deleted). Fusion proteins
comprising such 287
sequences may be used. All of these forms of 287, and more particularly those
which retain the
immunogenicity of wild-type 287 proteins, fall within the meaning of `287' as
used herein.
Another particularly preferred protein antigen from serogroup B N.meningitidis
is protein
`961', also known as 'NadA' [16]. This protein may be used in a wild-type form
[e.g. GenBank
accession gi:7227256; alleles of 961 are disclosed in ref. 17] but derivatives
of the wild-type protein
may be used. For instance, proteins having 50% or more sequence identity (e.g.
60%, 70%, 80%,
90%, 95%, 99% or more) to gi:7227256 may be used. Proteins comprising
truncation or deletion
variants of the protein may be used, such as those disclosed in references 10
to 12 ('961c' in
particular, which lacks the C-terminal membrane anchor). Fusion proteins
comprising such 961
sequences may be used. All of these forms of 961, and particularly those which
retain the
immunogenicity of wild-type 961 proteins, fall within the meaning of `961' or
'NadA' as used
herein.
Other preferred protein antigens are protein `741' and protein `ORF46.1', and
proteins
`ORF1', `ORF4', `ORF25', `ORF40', `ORF83', `NMB1343', `230', `233', `292',
`594', `687',
2
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
`736', `907', `919', `936', `953', and `983'. Other preferred protein antigens
are the hybrid proteins
disclosed in references 10 to 12, particularly those comprising one or more
of. a 287 protein, a 953
protein, a 936 protein and/or a 741 protein.
Protein antigens may be derived from any strain of N.meningitidis. It is
preferred to use
antigens from strains 2996, MC58, 95N477 and 394/98.
As well as strain variants, single or multiple conservative amino acid
substitutions may be
made with altering the immunogenicity of antigens used according to the
present invention.
In addition to or in place of protein antigens, nucleic acid encoding a
protein antigen may be
included within compositions of the invention. The nucleic acid will be
expressed in vivo once
administered to a mammalian recipient and the protein antigen will be
produced. Such nucleic acid
immunization is well known [e.g. refs. 18 to 23 etc.]. The nucleic acid will
typically be a DNA
plasmid.
A preferred saccharide antigen derived from serogroup C N.fneningitidis is the
oligosaccharide conjugate used in MenjugateTM [24, 25], which contains 12 to
22 monosaccharide
units from the serogroup C capsular polysaccharide.
A preferred saccharide antigen derived from serogroup A is an oligosaccharide
in which one
or more of the hydroxyl groups on the constituent monosaccharide units has
been replaced by a
blocking group [26].
Further oligosaccharide antigens from serogroups A, W135 and Y are disclosed
in reference
27.
The composition of the invention may comprise more than one Neisserial
antigen. Where
saccharides from both serogroups A and C of N.meningitidis are included, it is
preferred that the ratio
(w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g. 2:1, 3:1,
4:1, 5:1, 10:1 or higher).
The composition of the invention is preferably an immunogenic composition or
vaccine.
Such compositions comprise an immunologically effective amount of the antigen.
By
`immunologically effective amount', it is meant that the administration to an
individual of a
composition of the invention comprising that amount of antigen (either in a
single dose or as part of a
series) is effective for raising a therapeutic or prophylactic immune
response. This amount varies
depending upon the health and physical condition of the individual to be
treated, age, the taxonomic
group of individual to be treated (e.g. non-human primate, primate, etc.), the
capacity of the
individual's immune system to synthesise antibodies, the degree of protection
desired, the
formulation of the vaccine, the treating physician's assessment of the medical
situation, and other rel-
evant factors. The amount may fall in a relatively broad range that can be
determined through routine
trials. Antigens will typically be present at a concentration of at least 1
g/ml each.
3
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
Dosage treatment may be a single dose or a multiple dose schedule (e.g.
including booster
doses).
The CpG oligonucleotide
CpG oligonucleotides are known for use as vaccine adjuvants [e.g. ref. 28] and
they induce
strong Thl immune responses. They are useful as parenteral and mucosal
adjuvants [29].
The CpG oligonucleotide used according to the present invention is a nucleic
acid which
includes at least one CG dinucleotide i.e. a cytosine nucleotide followed by a
guanosine nucleotide.
The oligonucleotide may contain multiple CG dinucleotides.
A CG sequence in the oligonucleotide may be flanked by two purines at the 5'
side and two
pyrimidines at the 3' side i.e. RRCGYY.
Cytosine nucleotides in the CpG oligonucleotide may be methylated, but it is
preferred that
they should be unmethylated.
The cytosine and guanosine nucleotides are preferably deoxynucleotides and the
nucleic acid
is preferably DNA. In order to enhance nuclease resistance, the
oligonucleotide may comprise a
modified backbone, such as a phosphorothioate backbone. As an alternative to
using DNA, it is
possible to use PNA (peptide nucleic acid). In addition, the oligonucleotides
can comprise
substitutions of the sugar moieties and nitrogenous base moieties.
The oligonucleotide preferably comprises between about 6 and about 100
nucleotides, more
preferably between about 8 and about 50 nucleotides, most preferably between
about 10 and about 40
nucleotides.
Oligonucleotides comprising at least one CG dinucleotide can conveniently be
prepared
using conventional oligonucleotide synthesis.
Examples of CpG oligonucleotide adjuvants are found in references 30 to 55.
The biodegradable polymer microparticle
Biodegradable polymer microparticles are known for use as vaccine adjuvants
[e.g. ref. 56].
They are useful as parenteral and mucosal adjuvants.
As well as being biodegradable, the polymer used to make the microparticles
will generally
be sterilizable and non-toxic (biocompatible). Suitable biodegradable polymers
are readily
commercially available and include those derived from polyhydroxybutyric acid;
polycaprolactone;
polyorthoester; polyanhydride; poly(hydoxybutyrate); and a poly(a-hydroxy
acid). Preferred
polymers are formed from one or more poly(a-hydroxy acid) e.g. poly(L-
lactide), poly(D,L-lactide),
copolymers of D,L-lactide and glycolide (such as poly(D,L-lactide-co-
glycolide), or a copolymer of
D,L-lactide and caprolactone. Microparticles formed from poly(D,L-lactide-co-
glycolide) ('PLG')
are preferred.
4
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
These polymers are available in a variety of molecular weights, and the
appropriate
molecular weight for a given antigen can readily be determined. For poly(L-
lactide), a suitable
molecular weight will be on the order of about 2000 to 250,000. For PLG,
suitable molecular weights
will generally range from about 10,000 to about 200,000, preferably about
15,000 to about 150,000,
and most preferably about 50,000 to about 100,000.
For PLG microparticles, a variety of lactide:glycolide ratios may be used and
the ratio is
largely a matter of choice, depending in part on the co-administered antigen
and the rate of
degradation desired. For example, a 50:50 PLG polymer, containing 50% D,L-
lactide and 50%
glycolide, will provide a fast resorbing copolymer while 75:25 PLG degrades
more slowly, and 85:15
and 90:10, even more slowly, due to the increased lactide component. A
suitable ratio of
lactide:glycolide is easily determined based on the nature of the antigen and
disorder in question.
Moreover, mixtures of microparticles with varying lactide:glycolide ratios
will find use in the
formulations in order to achieve the desired release kinetics for a given
antigen and to provide for
both a primary and secondary immune response. Degradation rate of the
microparticles of the present
invention can also be controlled by such factors as polymer molecular weight
and polymer
crystallinity.
The term `microparticle' as used herein, refers to a particle of about 100 nm
to about 150 m
in diameter, more preferably about 200 nm to about 30 pm in diameter, and most
preferably about
500 nm to about 10 m in diameter. Preferably, the microparticle will be of a
diameter that permits
parenteral administration without occluding needles and capillaries.
Microparticle size is readily
determined by techniques well known in the art, such as photon correlation
spectroscopy, laser
diffractometry and/or scanning, electron microscopy. The term `microparticle'
includes
`nanoparticles' [57] within its scope. Preferred microparticles are
microspheres, although lamellar
particles [58] may also be used.
Microparticles may be prepared using any of several methods well known in the
art [e.g. ref.
59]. For example, double emulsion/solvent evaporation techniques [e.g. refs.
60 & 61] can be used to
form the microparticles. These techniques involve the formation of a primary
emulsion consisting of
droplets of polymer solution containing the antigen (if antigen is to be
entrapped in the
microparticle), which is subsequently mixed with a continuous aqueous phase
containing a particle
stabilizer/surfactant.
More particularly, a water-in-oil-in-water (w/o/w) solvent evaporation system
can be used to
form the microparticles, as described in references 62, 63 and 64. In this
technique, the particular
polymer is combined with an organic solvent, such as ethyl acetate,
dimethylchloride (also called
methylene chloride and dichloromethane), acetonitrile, acetone, chloroform,
and the like. The
polymer will be provided in about a 2-15% solution, in organic solvent. An
approximately equal
amount of an antigen solution (e.g. in water) is added and the polymer/antigen
solution emulsified
5
CA 02462646 2011-02-22
using e.g. a homogenizer. The emulsion is then combined with a larger volume
of an aqueous
solution of an emulsion stabilizer such as polyvinyl alcohol (PVA) or
polyvinyl pyrrolidone. The
emulsion stabilizer is typically provided in about a 2-15% solution, more
typically about a 4-10%
solution. The mixture is then homogenized to produce a stable w/o/w double
emulsion. Organic
solvents are then evaporated.
The formulation parameters can be manipulated to allow the preparation of
small (<5 Am) and
large (>301tm) microparticles [e.g. 63, 65]. For example, reduced agitation
results in larger
microparticles, as does an increase in internal phase volume. Small particles
are produced by low
aqueous phase volumes with high concentrations of PVA.
Microparticles can also be formed using spray-drying and coacervation [e.g.
refs. 66, 67 &
68]; air-suspension coating techniques, such as pan coating and Wurster
coating [69, 70]; ionic
gelation [71].
Prior to use of the microparticles, antigen content is generally determined so
that an
appropriate amount of the microparticles may be delivered to the subject in
order to elicit an
adequate immune response.
Antigen content of the microparticles can be determined according to methods
known in the
art, such as by disrupting the microparticles and extracting entrapped
antigen. For example,
microparticles can be dissolved in dimethylchloride and the protein extracted
into distilled water [e.g.
refs. 72, 73, 74]. Alternatively, microparticles can be dispersed in 0.1 M
NaOH containing 5% (w/v)
SDS. The sample is agitated, centrifuged and the supernatant assayed for
antigen using an
appropriate assay [75].
Antigen and/or CpG-oligonucleotides can be located within or on the
microparticles.
Entrapment will generally be achieved by having the antigen/oligonucleotide
present during
formation of the microparticles, whereas surface adsorption is achieved by
adding
antigen/oligonucleotide to pre-formed microparticles.
One method for adsorbing antigen/oligonucleotide onto prepared microparticles
is as follows.
Microparticles are rehydrated and dispersed to an essentially monomeric
suspension of
microparticles using dialyzable anionic or cationic detergents. Useful
detergents include, but are not
limited to, any of the various N-methylglucamides (known as MEGAs), such as
heptanoyl-N-
methylglucamide (MEGA-7), octanoyl-N-methylglucamide (MEGA-8), nonanoyl-N-
methylglucamide (MEGA-9), and decanoyl-N-methyl-glucamide (MEGA-10); cholic
acid; sodium
cholate; deoxycholic acid; sodium deoxycholate; taurocholic acid; sodium
taurocholate;
taurodeoxycholic acid; sodium taurodeoxycholate; 3-[(3-
cholamidopropyl)dimethylammonio]-1-
propane-sulfonate (CHAPS); N-octylglucoside; 3-[(3-cholamidopropyl)
dimethylammonio]-2-
hydroxy-l-propane-sulfonate (CHAPSO); N-dodecyI-N,N-dimethyl-3-ammonio-l-
propane-sulfonate
(ZWITTERGENT* 3-12); N,N-bis-(3-D-gluconeamidopropyl)-deoxycholamide (DEOXY-
Trade-mark 6
CA 02462646 2011-02-22
BIGCHAP); sucrose monolaurate; glycocholic acid/sodium glycocholate;
laurosarcosine (sodium
salt); glycodeoxycholic acid/sodium glycodeoxycholate; sodium dodceyl sulfate
(SDS); and
hexadecyltrimethylammonium bromide (CTAB); dodecyltrimethylammonium bromide;
hexadecyltrimethyl-ammonium bromide; tetradecyltrimethylammonium bromide;
benzyl
dimethyldodecylammonium bromide; benzyl dimethyl-hexadecylammonium chloride;
benzyl
dimethyltetra-decylammonium bromide. The above detergents are commercially
available. Various
cationic lipids known in the art can also be used as detergents [76, 77].
The microparticle/detergent mixture is then physically ground e.g. using a
ceramic mortar
and pestle, until a smooth slurry is formed. An appropriate aqueous buffer,
such as phosphate
buffered saline (PBS) or Tris buffered saline, is then added and the resulting
mixture sonicated or
homogenized until the microparticles are fully suspended. The
antigen/oligonucleotide is then added
to the microparticle suspension and the system dialyzed to remove detergent.
The polymer
microparticles and detergent system are preferably chosen such that the
antigen/oligonucleotide will
adsorb to the microparticle surface while still maintaining activity . The
resulting microparticles
containing surface adsorbed antigen/oligonucleotide may be washed free of
unbound
antigen/oligonucleotide and stored as a suspension in an appropriate buffer
formulation, or
lyophilized with the appropriate excipients, as described further below.
The antigen/CpG/microparticle combination
Various physical relationships are possible between the three basic components
of the
compositions of the invention. These arise because the microparticles have an
internal volume and a
surface, either of which may be used to locate the CpG-oligonucleotide and/or
the antigen.
Thus the antigen may be entrapped within microparticles, it may be adsorbed to
microparticles, or it may be in simple admixture with the microparticles
without entrapment or
adsorption. Adsorption is preferred.
Similarly, the CpG-oligonucleotide may be entrapped within microparticles, it
may be
adsorbed to microparticles, or it may be in simple admixture with the
microparticles. Adsorption can
be achieved using detergents such as CTAB.
The CpG-oligonucleotide and the antigen may both have the same physical
relationship to
the microparticles as each other, or they may be different. Likewise the CpG-
oligonucleotide and the
antigen may be adsorbed onto the same microparticle or the CpG-oligonucleotide
and the antigen
may be adsorbed onto different microparticles. All possible combinations are
encompassed within
the present invention:
CpG-oligonucleotide
Entrapped Adsorbed Mixed
Entrapped Yes Yes Yes
Adsorbed Yes Yes Yes
7
Trade-mark
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
I I Mixed Yes Yes Yes
Compositions of the invention may include mixtures of the above e.g. some
microparticles within the
composition have entrapped antigen and some have adsorbed antigen.
Pharmaceutical compositions
For pharmaceutical use, compositions of the invention will generally comprise
a
pharmaceutically acceptable carrier. This gives a pharmaceutical composition
of the invention.
A pharmaceutically acceptable carrier can be any substance that does not
itself induce the production
of antibodies harmful to the patient receiving the composition, and which can
be administered
without undue toxicity. Suitable carriers can be large, slowly-metabolised
macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid
copolymers, and inactive virus particles. Such carriers are well known to
those of ordinary skill in the
art. Pharmaceutically acceptable carriers can include liquids such as water,
saline, glycerol and
ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH
buffering substances, and
the like, can also be present in such vehicles. Liposomes are suitable
carriers. A thorough discussion
of pharmaceutical carriers is available in ref. 78.
Compositions of the invention may be prepared in various forms. For example,
the
compositions may be prepared as injectables, either as liquid solutions or
suspensions. Solid forms
suitable for solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The
composition may be prepared for topical administration e.g. as an ointment,
cream or powder. The
composition be prepared for oral administration e.g. as a tablet or capsule,
or as a syrup (optionally
flavoured). The composition may be prepared for pulmonary administration e.g.
as an inhaler, using
a fine powder or a spray. The composition may be prepared as a suppository or
pessary. The
composition may be prepared for nasal, aural or ocular administration e.g. as
drops, as a spray, or as
a powder [e.g. 79].
The pharmaceutical composition is preferably sterile. It is preferably pyrogen-
free. It is
preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7.
The pharmaceutical composition may be lyophilised.
The invention also provides a delivery device containing a pharmaceutical
composition of the
invention. The device may be, for example, a syringe.
Medical treatments and uses
Compositions of the invention may be used therapeutically (i.e. to treat an
existing Neisserial
infection) or prophylactically (i.e. to prevent future Neisserial infection).
The invention provides a composition of the invention for use as a medicament.
8
CA 02462646 2011-02-22
The invention also provides a method for raising an antibody response in a
mammal,
comprising administering a pharmaceutical composition of the invention to the
mammal. The
antibody response is preferably an IgA or IgG response and it is preferably
bactericidal.
The invention also provides a method for treating a mammal suffering from a
Neisserial
infection and/or disease, comprising administering to the patient a
pharmaceutical composition of the
invention.
The invention also provides a method for protecting a mammal against a
Neisserial infection
and/or disease, comprising administering to the mammal a pharmaceutical
composition of the
invention.
The invention also provides the use of (a) a Neisserial antigen, (b) a CpG
oligonucleotide,
and (c) a biodegradable polymer microparticle, in the manufacture of a
medicament for preventing or
treating disease and/or infection in an mammal.
The mammal is preferably a human. The human may be an adult or, preferably, a
child.
Compositions of the invention are particularly useful for immunising children
and teenagers.
The uses and methods of the invention are particularly useful for
treating/protecting against
infections of N.ineningitidis. The uses and methods are particularly useful
for preventing/treating
diseases including bacterial meningitis.
Efficacy of therapeutic treatment can be tested by monitoring Neisserial
infection after
administration of the composition of the invention. Efficacy of prophylactic
treatment can be tested
by monitoring anti-Neisseria immune responses after administration of the
composition.
Compositions of the invention will generally be administered directly to a
patient. Direct delivery
may be accomplished by parenteral injection (e.g. subcutaneously,
intraperitoneally, intravenously,
intramuscularly, or to the interstitial space of a tissue), or by rectal,
oral, vaginal, topical,
trarisdermal, ocular, nasal, aural, or pulmonary administration. Injection or
intranasal administration
is preferred.
Dosage treatment can be a single dose schedule or a multiple dose schedule.
Further components
Compositions of the invention may include adjuvants in addition to CpG-
oligonucleotides
and polymer microparticles. Preferred further adjuvants include, but are not
limited to: (A)
aluminium compounds (e.g. aluminium hydroxide, aluminium phosphate, aluminium
hydroxyphosphate, oxyhydroxide, orthophosphate, sulphate etc. [e.g. see
chapters 8 & 9 of ref. 13]),
or mixtures of different aluminium compounds, with the compounds taking any
suitable form (e.g.
gel, crystalline, amorphous etc.), and with adsorption being preferred; (B)
MF59 (5% Squalene, 0.5%
Tween 80, and 0.5% Span 85, formulated into submicron particles using a
microfluidizer) [see
Chapter 10 of 13; see also ref. 80]; (C) liposomes [see Chapters 13 and 14 of
ref. 13]; (D) ISCOMs
* Trade-mark 9
CA 02462646 2011-02-22
[see Chapter 23 of ref. 13], which may be devoid of additional detergent [81];
(E) SAF, containing
10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP,
either
microfluidized into a submicron emulsion or vortexed to generate a larger
particle size emulsion [see
Chapter 12 of ref. 13]; (F) RibiTm adjuvant system (RAS), (Ribi Imrnunochem)
containing 2%
Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from
the group consisting
of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall
skeleton (CWS),
preferably MPL + CWS (DetoxTM); (G) saponin adjuvants, such as QuilA or QS21
[see Chapter 22
of ref. 13], also known as StimulonTM [82]; (H) chitosan [e.g. 83]; (I)
complete Freund's adjuvant
(CFA) and incomplete Freund's adjuvant (1FA); (J) cytokines, such as
interleukins (e.g. IL-1, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-y),
macrophage colony stimulating
factor, tumor necrosis factor, etc. [see Chapters 27 & 28 of ref. 13]; (K)
monophosphoryl lipid A
(MPL) or 3-0-deacylated MPL (3dMPL) [e.g. chapter 21 of ref. 13]; (L)
combinations of 3dMPL
with, for example, QS21 and/or oil-in-water emulsions [84]; (M) a
polyoxyethylene ether or a
polyoxyethylene ester [85]; (N) a polyoxyethylene sorbitan ester surfactant in
combination with an
octoxynol [86] or a polyoxyethylene alkyl ether or ester surfactant in
combination with at least one
additional non-ionic surfactant such as an octoxynol [87]; (N) a particle of
metal salt [88]; (0) a
saponin and an oil-in-water emulsion [89]; (P) a saponin (e.g. QS21) + 3dMPL-+
IL-12 (optionally +
a sterol) [90]; (Q) E.coli heat-labile enterotoxin ("LT"), or detoxified
mutants thereof, such as the
K63 or R72 mutants [e.g. Chapter 5 of ref. 91]; (R) cholera toxin ("CT"), or
detoxified mutants
thereof [e.g. Chapter 5 of ref. 91]; (S) double-stranded RNA and (T) other
substances that act as
immunostimulating agents to enhance the effectiveness of the composition [e.g.
see Chapter 7 of ref.
13]. Alum (especially aluminium phosphate and/or hydroxide) and MF59 are
preferred further
adjuvants for parenteral immunisation. Mutant toxins are preferred mucosal
adjuvants.
Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-
acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-
D-isoglutaminyl-
L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine
MTP-PE), etc.
As well as Neisserial antigen(s), the composition may comprise further
antigenic
components. Antigens which can be included in the composition of the invention
include:
- antigens from Helicobacter pylori such as CagA [92 to 95], VacA [96, 97],
NAP [98, 99,
100], HopX [e.g. 101], HopY [e.g. 1011 and/or urease.
- an outer-membrane vesicle (OMV) preparation from N.meningitidis serogroup B,
such as
those disclosed in refs. 102, 103, 104, 105 etc.
- a saccharide antigen from Streptococcus pneumoniae [e.g. 106, 107, 108].
- an antigen from hepatitis A virus, such as inactivated virus [e.g. 109,
110].
- an antigen from hepatitis B virus, such as the surface and/or core antigens
[e.g. 110, 111].
- an antigen from hepatitis C virus [e.g. 112].
Trade-mark
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
- an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and
filamentous
haemagglutinin (FHA) from B.pertussis, optionally also in combination with
pertactin and/or
agglutinogens 2 and 3 [e.g. refs. 113 & 114].
- a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of ref.
115] e.g. the CRM197
mutant [e.g. 116].
- a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of ref. 115].
- a saccharide antigen from Haemophilus influenzae B [e.g. 23].
- an antigen from Chlamydia pneumoniae [e.g. 117, 118, 119, 120, 121, 122,
123].
- an antigen from Chlaniydia trachomatis [e.g. 124].
- an antigen from Porphyromonas gingivalis [e.g. 125].
- polio antigen(s) [e.g. 126, 127] such as IPV or OPV.
- rabies antigen(s) [e.g. 128] such as lyophilised inactivated virus [e.g.
129, RabAvertTM].
- measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref.
115].
- antigen(s) from influenza virus [e.g. chapter 19 of ref. 115], such as the
haemagglutinin
and/or neuraminidase surface proteins
- antigen(s) from a paarmyxovirus such as respiratory syncytial virus (RSV
[130, 131]) and/or
parainfluenza virus (PIV3 [132]).
- an antigen from Moraxella catarrhalis [e.g. 133].
- an antigen from Streptococcus agalactiae (group B streptococcus) [e.g. 134,
135].
- an antigen from Streptococcus pyogenes (group A streptococcus) [e.g. 135,
136, 137].
- an antigen from Staphylococcus aureus [e.g. 138].
- an antigen from Bacillus anthracis [e.g. 139, 140, 141].
- an antigen from a virus in the flaviviridae family (genus flavivirus), such
as from yellow
fever virus, Japanese encephalitis virus, four serotypes of Dengue viruses,
tick-borne
encephalitis virus, West Nile virus.
- a pestivirus antigen, such as from classical porcine fever virus, bovine
viral diarrhoea virus,
and/or border disease virus.
- a parvovirus antigen e.g. from parvovirus B19.
- a prion protein (e.g. the CJD prion protein)
- an amyloid protein, such as a beta peptide [142]
- a cancer antigen, such as those listed in Table 1 of ref. 143 or in tables 3
& 4 of ref. 144.
The composition may comprise one or more of these further antigens.
Toxic protein antigens may be detoxified where necessary (e.g. detoxification
of pertussis
toxin by chemical and/or genetic means [114]).
Where a diphtheria antigen is included in the composition it is preferred also
to include
tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is
included it is preferred
11
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
also to include diphtheria and pertussis antigens. Similarly, where a
pertussis antigen is included it is
preferred also to include diphtheria and tetanus antigens.
Antigens are preferably adsorbed to an aluminium salt.
Antigens in the composition will typically be present at a concentration of at
least 1 g/ml
each. In general, the concentration of any given antigen will be sufficient to
elicit an immune
response against that antigen.
As an alternative to using proteins antigens in the composition of the
invention, nucleic acid
encoding the antigen may be used. Protein components of the compositions of
the invention may thus
be replaced by nucleic acid (preferably DNA e.g. in the form of a plasmid)
that encodes the protein.
Definitions
The term "comprising" means "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.
References to a percentage sequence identity between two amino acid sequences
means that,
when aligned, that percentage of amino acids are the same in comparing the two
sequences. This
alignment and the percent homology or sequence identity can be determined
using software programs
known in the art, for example those described in section 7.7.18 of reference
145. A preferred
alignment is determined by the Smith-Waterman homology search algorithm using
an affine gap
search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM
matrix of 62. The
Smith-Waterman homology search algorithm is taught in reference 146.
MODES FOR CARRYING OUT THE INVENTION
Parenteral prime & mucosal boost with Neisseria meningitidis serogroup B
antigen
Reference 6 discloses a protein from serogroup B N.meningitidis called `287'.
References 10
to 12 disclose ways of improving its expression. One way involves deleting the
N-terminus of the
protein up to and including the six repeated glycine residues. This protein is
referred to as 'AG287'.
Mice were primed and boosted with MenB AG287 antigen (201tg/dose) from strain
2996,
formulated for intramuscular (IM) administration by adsorption to PLG
microparticles, with or
without CpG oligonucleotide (also adsorbed to the microparticles). An
additional formulation for
intranasal (IN) administration used LT-K63 adjuvant. Mice received either 3 IM
doses or 2 IM then 2
IN doses (doses at: day 0; day 28; day 84; and, optionally, day 98).
Antibody GMT 2 weeks after
Group Formulation Route Dose dose 2 Dose 3 dose 4
1 PLG/287 IM 1, 2, 3 10,729 2,853 -
2 PLG/287 + PLG/CpG IM 1, 2, 3 15,673 4,163 -
3 PLG/287 IM 1, 2 9,064 7,948 9,412
12
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
287 + LT-K63 IN 3, 4
PLG/287 + PLG/CpG IM 1,2 4 34,891 15,167 16,556
287 + LT-K63 IN 3,4
Thus the inclusion of CpG-oligonucleotide enhanced antibody titers against
intramuscularly
administered MenB protein 287 (compare groups 1 & 2). Titers could be enhanced
by replacing a
third intramuscular dose with two intranasal doses (compare groups 1 & 3). The
CpG enhancement
was also seen in the intramuscular/intranasal regime (compare groups 3 & 4).
Comparison of adjuvants for MenB protein 287
AG287 was formulated with various adjuvants and administered to mice. Sera
from the mice
were assessed using the bactericidal antibody (BCA) assay and titers were as
follows:
Adjuvant BCA post-2 BCA post-3
Freund's adjuvant 2048 8192
Alum <4 256
Alum + CpG oligonucleotide 256 4096
MF59 <4 <4
CpG oligonucleotide <4 128
PLG microparticles (adsorbed) 8 1024
PLG microparticles (adsorbed) + CpG 2048 16384
CpG-oligonucleotide was thus only mildly effective as an adjuvant, almost
comparable to
alum. The PLG microparticles were more effective than both alum and CpG, but
not as effective as
Freund's adjuvant. In marked contrast, however, the mixture of CpG and PLG
matched the
adjuvanticity of Freund's adjuvant at the post-second immunization stage and
exceeded Freund's
adjuvant post-third immunization.
Enhancement of PLG adjuvanticity by using CpG was also seen in a separate
study (02-
0279):
Adjuvant GMT post-2 GMT post-3
MF59 6967 13417
PLG microparticles (adsorbed) 7070 11367
PLG microparticles (adsorbed) + CpG 15099 26833
Effect of adsorption on adjuvanticity
The effect of adsorption on adjuvanticity was studied. Protein AG287 was
either adsorbed
onto PLG microparticles using DSS surfactant or SDS or was simply mixed with
the particles.
Immunisations were performed on days 0, 21 and 35 and titers were assessed on
days 35 and 49.
20, Results were as follows:
13
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
Antibody titer 2 weeks after
Formulation BCA dose 2 Dose 3
CpG + 287 adsorbed on PLG (DSS) 4096 45817 67921
CpG + 287 adsorbed on PLG (SDS) 4096 39730 29911
CpG + 287 + PLG (no adsorption) <16 62 1065
DSS + 287 adsorbed on alum <16 1209 1249
CpG + 287 adsorbed on Alum 1024 4054 12236
287 adsorbed on alum 128 646 2454
The adjuvanticity of CpG and microparticle mixtures for AG287 is thus optimal
when the
antigen is adsorbed to microparticles.
Reference 6 discloses a protein from serogroup B N.ineningitidis called `961'
(now known as
'NadA' [16,17]). References 10 to 12 disclose ways of improving the expression
of NadA. One way
involves deleting the C-terminus of the protein to remove its membrane anchor
(i.e. remove amino
acids 351-405 for strain 2996), as well as natural removal of its leader
peptide. This protein is
referred to as '961c'. The effect of adsorption on PLG adjuvanticity when co-
administered with CpG
was studied for 961c, as described above for 287:
Antibody titer 2
weeks
Formulation BCA after dose 3
961 adsorbed on PLG (SDS) 2048 20661
961 + PLG (no adsorption) 256 1706
287 adsorbed on PLG 4096 63057
287 adsorbed on PLG + 961 soluble 4096 287:86052;961:1924
287 adsorbed on PLG + 961 adsorbed on PLG 8192 287:107142;961:11717
287 (not adsorbed) + 961 (not adsorbed) + `blank' 1024 287:1266;961:145
PLG
287 (adsorbed) + 961 (adsorbed) + `blank' PLG 8192 287: 78176; 961: 20876
As for AG287, therefore, the adjuvanticity of CpG and microparticle mixtures
for 961c is
optimal when the antigen is adsorbed to microparticles. This is true for the
antigen on its own and the
antigen when combined with AG287.
For both AG287 and 961c, therefore, singly and combined, the best
adjuvanticity for CpG
and PLG mixtures is seen when the antigens are adsorbed onto the PLG
microparticles.
14
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
PLG, CpG, alum and MF59
Various combinations of PLG, CpG and alum were tested for protein AG287,
expressed as a
His-tagged product. Serum bactericidal titers after three immunisations were
as follows:
Adjuvant Titer
Alum 2048
Alum + CpG 32768
MF59 8192
MF59 + CpG 32768
PLG (antigen adsorbed to PLG) 1024
PLG + CpG (antigen and CpG both adsorbed to PLG) 4096
PLG + MF59 (antigen adsorbed to PLG) 2048
PLG + MF59 + CpG (antigen adsorbed to PLG) 8192
Complete Freund's 32768
PLG + Complete Freund's (antigen adsorbed to PLG) 2048
Similar experiments were performed and results were as follows:
Adjuvant Titer
PLG (antigen adsorbed to PLG) 1024
PLG + CpG (antigen adsorbed to PLG) 16384
PLG + CpG (antigen and CpG both adsorbed to PLG) 16384
PLG + alum (antigen adsorbed to PLG) 1024
PLG + alum + CpG (antigen adsorbed to PLG) 16384
PLG + alum + CpG (antigen and CpG both adsorbed to PLG) 8192
PLG + MF59 (antigen adsorbed to PLG) 4096
PLG + MF59 + CpG (antigen adsorbed to PLG) 16384
Alum (antigen adsorbed to alum) 256
CpG 128
Alum + CpG 1024
Alum + CpG + PLG (antigen adsorbed to alum; CpG adsorbed to PLG) 4096
CpG + PLG (CpG adsorbed on PLG; antigen not adsorbed) 64
Thus MF59 and alum can further enhance efficacy of CpG/PLG mixtures,
adsorption of CpG
to PLG microparticles is not necessary for adjuvanticity, but adsorption of
antigen to microparticles
is again seen to be optimal.
Antigen mixtures
The effect of adsorption on adjuvanticity was studied for proteins AG287 and
961c, singly
and in combination. Antibody titers after three doses were as follows:
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
Antibody GMT against
Formulation 287 961
CpG + 961 adsorbed on PLG - 20661
CpG + 961 + PLG (no adsorption) - 1706
CpG + 961 + 287 adsorbed on PLG 86052 1924
CpG + 961 adsorbed on PLG + 287 adsorbed on PLG 107142 11717
CpG + 287 adsorbed on PLG 63057 -
CpG + 287 & 961 co-adsorbed on PLG 57306 6251
CpG + 961 adsorbed on PLG + 287 adsorbed on PLG + PLG 78176 20876
287 + 961 + PLG (no adsorption of antigens) 1266 145
As for AG287, therefore, the adjuvanticity of CpG and microparticle mixtures
for protein
961c is optimal when the antigen is adsorbed to microparticles.
Further combinations of adjuvants with PLG microparticles were tested for
proteins AG287
and 961c. The CpG was either soluble or was adsorbed to PLG microparticles.
Results were as
follows:
GMT against
Formulation + PLG microparticles BCA 287 961
287 (adsorbed on PLG) + 961 (adsorbed on PLG) 256 5719 2412
287 (adsorbed on PLG) + 961 (adsorbed on PLG) + CpG 512 17553 8627
287 (adsorbed on PLG) + 961 (adsorbed on PLG) + CpG (adsorbed on PLG) 1024
16906 6720
287 (adsorbed on PLG) + 961 (adsorbed on PLG) + MF59 64 4636 3969
287 (adsorbed on PLG) + 961 (adsorbed on PLG) + MF59 + CpG 2048 23642 48446
Similar work was performed on groups of 10 CD-1 mice, using 201tg per PLG-
adsorbed
antigen per IM dose (days 0, 21 and 35). Where CpG was present, it was given
at 10 g per dose.
ELISA titers (GMT) were calculated as the reciprocal serum dilution giving
OD450. 0.5, and sera
were tested for both antigens. Serum bactericidal activity titers (SBA) are
calculated as the reciprocal
serum dilution killing 50% of target bacteria, and sera were tested for
activity against 2996 strain and
against MC58, a heterologous strain. Titers at day 49 (2 weeks post-third
dose) were as follows:
287 961 Extra adjuvant GMT SBA
287 961 2996 MC58
X - - 8375 - 512 <4
X - Soluble CpG 33736 - 1024 128
X - PLG-adsorbed CpG 32058 - 1024 64
X - - 3818 nd nd
X Soluble CpG - 14149 2048 <4
- X PLG-adsorbed CpG - 18526 2048 <4
16
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
X X - 13557 2476 nd nd
X X Soluble CpG 21664 6557 8192 64
X X PLG-adsorbed CpG 27259 7510 2048 128
X X Soluble CpG + MF59 27981 26826 2048 256
Control: soluble 287 with CFA 37889 - 1024 <32
Control: soluble 961 with CFA - 50453 4096 <4
Control: soluble 287 and 961 with CFA 1678 27069 512 <32
Reference 12 discloses a combination of three proteins which, between them,
include five
different N.nzeningitidis antigens: (1) 96102996; (2) AG287NZ-9532996; and (3)
9362996-AG741MC58=
The antigen mixture was tested in reference 12 using aluminum hydroxide
adjuvant. According to the
present invention, the antigen mixture is adjuvanted by adsorption to a
biodegradable polymer
microparticle plus a CpG oligonucleotide. Titers after the third dose were as
follows:
ELISA GMT SBA (against seven strains)
Immunisation 961 287 741 953 2996 MC58 BZ133 394/98 NGH38 F6124 44/76
(1) 961 on alum 12346 - - - 4096 <4 <4 <4 <4 64 <4
(2) 287-953 on alum - 6415 - 585 1024 1024 256 1024 4096 256 1024
(3) 936-741 on alum - - 10625 - <4 32768 16384 1024 128 16384 32768
(1), (2) & (3) on alum 42302 18206 33881 4549 8192 32768 32768 2048 4096 32768
65536
(1) 961 on PLG 14185 - - - 2048 4 <4 <4 16 256 <4
(2) 287-953 on PLG - 43515 - 478 2048 128 2048 2048 8192 4096 128
(3) 936-741 on PLG - - 16150 - <4 32768 16384 1024 512 8192 262144
(1),(2) & (3) on PLG 6735 24304 13801 1214 4096 65536 32768 2048 4096 32768
65536
(1), (2) & (3) on PLG + CpG 10896 40697 26966 2301 8192 262144 65536 4096 8192
32768 262144
Compared to the aluminum adjuvant used in reference 12, the PLG+CpG mixture
leads to
lower overall antibody titers (except for protein 287) but, importantly, gives
higher bactericidal titers
against a wide range of strains. Although absolute titers are lower,
therefore, the adjuvant of the
invention therefore advantageously shifts antibody production towards
bactericidal antibodies.
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.
17
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
REFERENCES (the contents of which are hereby incorporated by reference)
[1] Parkhill et al. (2000) Nature 404:502-506.
[2] Tettelin et al. (2000) Science 287:1809-1815.
[3] W000/66791.
[4] W099/24578.
[5] W099136544.
[6] W099/57280.
[7] W000/22430.
[8] W000/66741.
[9] Pizza et al. (2000) Science 287:1816-1820.
[10] WO01/64920.
[11] WO01/64922.
[12] International patent application PCT/1802/03904.
[13] Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman,
Plenum Press
1995 (ISBN 0-306-44867-X).
[14] Jones (2001) Curr Opin Investig Drugs 2:47-49.
[15] W000/50075.
[16] Comanducci et al. (2002) J. Exp. Med. 195:1445-1454.
[17] International patent application PCT/1B02/03396.
[18] Strugnell et al. (1997) Immunol Cell Biol 75(4):364-369.
[19] Robinson & Torres (1997) Seminars in Immunol 9:271-283.
[20] Donnelly et al. (1997) Annu Rev Immunol 15:617-648.
[21] DNA Vaccination - Genetic Vaccination (eds. Koprowski et al.; 1998) ISBN
3540633928.
[22] Brunham et al. (2000) J Infect Dis 181 Suppl 3:S538-43.
[23] Svanholm et al. (2000) Scand J Immunol 51(4):345-53.
[24] Costantino et al. (1992) Vaccine 10:691-698.
[25] Costantino et al. (1999) Vaccine 17:1251-1263.
[26] UK patent applications 0207117.3 & 0220195.2
[27] International patent application PCT/1802/03191.
[28] McCluskie et al. (2001) Curr. Opin. Investig. Drugs 2:35-39.
[29] McCluskie et al. (2001) Crit. Rev. Immunol. 21:103-120.
[30] Krieg et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 12631-12636,
[31] Klinman et al. (1996), Proc. Natl. Acad. Sci. USA, 93, 2879-2883
[32] Weiner et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 10833-10837
[33] Chu et al. (1997) J. Exp. Med., 186, 1623-1631
[34] Brazolot-Millan et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 15553-15558
[35] Ballas et al. (1996) J. Immunol., 157, 1840-1845
[36] Cowdery et al. (1996) J. Immunol., 156, 4570-4575
18
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
[37] Halpern et al. (1996) Cell. Immunol., 167, 72-78
[38] Yamamoto et al. (1988) Jpn. J. Cancer Res., 79, 866-873
[39] Stacey et al. (1996) J. Iminunol., 157, 2116-2122
[40] Messina et al. (1991) J. bninunol.,147, 1759-1764
[41] Yi et al. (1996) J. bnrnunol., 157, 4918-4925
[42] Yi et al. (1996) J. bnrnunol., 157, 5394-5402
[43] Yi et al. (1998) J. Immunol., 160, 4755-4761
[44] Roman et al. (1997) Nat. Med., 3, 849-854
[45] Davis et al. (1998) J. Immunol.,160, 870-876
[46] Lipford et al. (1997) Eur. J. Immunol., 27, 2340-2344
[47] Moldoveanu et al. (1988) Vaccine, 16, 1216-1224
[48] Yi et al. (1998) J. Immunol., 160, 5898-5906
[49] W096/02555
[50] WO 98/16247
[51] W098/18810
[52] W098/40100
[53] W098/55495
[54] W098/37919a
[55] W098/52581
[56] Gupta et al. (1998) Adv Drug Deliv Rev 32:225-246.
[57] Ravi Kumar (2000) J Pharm Pharm Sci 3:234-258.
[58] Jabbal-Gill et al. (2001) Adv Drug Deliv Rev 51:97-111.
[59] Jain (2000) Biomaterials 21:2475-2490.
[60] U.S. Patent No. 3,523,907
[61] Ogawa et al. (1988) Chem. Pharm. Bull. 36:1095-1103.
[62] O'Hagan et al. (1993) Vaccine 11:965-969.
[63] Jeffery et al. (1993) Pharnr. Res. 10:362-368.
[64] WO 00/06133
[65] McGee et al. (1997) J Microencapsul. 14:197-210.
[66] Thomasin et al. (1996) J. Controlled Release 41:131ff
[67] U.S. Patent 2,800,457
[68] Masters, K. (1976) Spray Drying 2nd Ed. Wiley, New York
[69] Hall et al., (1980) The "Wurster Process" in Controlled Release
Technologies: Methods,
Theory, and Applications (A.F. Kydonieus, ed.), Vol. 2, pp. 133-154 CRC Press,
Boca Raton, Florida
[70] Deasy, P.B. (1988) Crit. Rev. Ther. Drug Carrier Syst. S(2):99-139
[71] Lim et al. (1980) Science 210:908-910.
[72] Cohen et al. (1991) Pharm. Res. 8:713ff.
19
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
[73] Eldridge et al. (1991) Infect. Imnaun. 59:2978ff.
[74] Eldridge et al. (1990) J. Controlled Release 11:205ff.
[75] O'Hagan et al. (1994) Int. J. Pharm. 103:37-45.
[76] Balasubramaniam et al. (1996) Gene Ther. 3:163-172.
[77] Gao & Huang (1995) Gene Ther. 2:7110-7122.
[78] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th
edition, ISBN:
0683306472.
[79] Almeida & Alpar (1996) J. Drug Targeting 3:455-467.
[80] W090/14837.
[81] W000/07621.
[82] W000/62800.
[83] W099/27960.
[84] European patent applications 0835318, 0735898 and 0761231.
[85] W099/52549.
[86] WO01/21207.
[87] WO01/21152.
[88] W000/23105.
[89] W099/11241.
[90] W098/57659.
[91] Del Giudice et al. (1998) Molecular Aspects of Medicine, vol. 19, number
1.
[92] Covacci & Rappuoli (2000) J. Exp. Med. 19:587-592.
[93] W093/18150.
[94] Covacci et al. (1993) Proc. Natl. Acad. Sci. USA 90: 5791-5795.
[95] Tummuru et al. (1994) Infect. Immun. 61:1799-1809.
[96] Marchetti et al. (1998) Vaccine 16:33-37.
[97] Telford et al. (1994) J. Exp. Med. 179:1653-1658.
[98] Evans et al. (1995) Gene 153:123-127.
[99] W096/01272 & W096/01273, especially SEQ ID NO:6.
[100] W097/25429.
[101] W098/04702.
[102] WO01/52885.
[103] Bjune et al. (1991) Lancet 338(8775):1093-1096.
[104] Fukasawa et al. (1999) Vaccine 17:2951-2958.
[105] Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333.
[106] Watson (2000) Pediatr Infect Dis J 19:331-332.
[107] Rubin (2000) Pediatr Clin North Ana 47:269-285, v.
[108] Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207.
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
[109] Bell (2000) Pediatr Infect Dis J 19:1187-1188.
[110] Iwarson (1995) APMIS 103:321-326.
[111] Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.
[112] Hsu et al. (1999) Clin Liver Dis 3:901-915.
[113] Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355.
[ 114] Rappuoli et al. (1991) TIBTECH 9:232-238.
[115] Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.
[116] Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.
[117] W002/02606.
[118] Kalman et al. (1999) Nature Genetics 21:385-389.
[119] Read et al. (2000) Nucleic Acids Res 28:1397-406.
[120] Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):5524-S527.
[121] W099/27105.
[122] W000/27994.
[123] W000/37494.
[124] W099/28475.
[125] Ross et al. (2001) Vaccine 19:4135-4142.
[126] Sutter et al. (2000) Pediatr Clin North Am 47:287-308.
[127] Zimmerman & Spann (1999) Ain Fain Physician 59:113-118, 125-126.
[128] Dreesen (1997) Vaccine 15 Suppl:S2-6.
[129] MMWR Morb Mortal Wkly Rep 1998 Jan 16;47(1):12, 19.
[130] Anderson (2000) Vaccine 19 Suppl 1:S59-65.
[131] Kahn (2000) Curr Opin Pediatr 12:257-262.
[132] Crowe (1995) Vaccine 13:415-421.
[133] McMichael (2000) Vaccine 19 Suppl 1:S101-107.
[134] Schuchat (1999) Lancet 353(9146):51-6.
[135] W002/34771.
[136] Dale (1999) Infect Dis Clin North Ain 13:227-43, viii.
[137] Ferretti et al. (2001) PNAS USA 98: 4658-4663.
[138] Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages 1218-
1219.
[139] J Toxicol Clin Toxicol (2001) 39:85-100.
[140] Demicheli et al. (1998) Vaccine 16:880-884.
[141] Stepanov et al. (1996) JBiotechnol 44:155-160.
[142] Ingram (2001) Trends Neurosci 24:305-307.
[143] Rosenberg (2001) Nature 411:380-384.
[144] Moingeon (2001) Vaccine 19:1305-1326.
21
CA 02462646 2004-04-02
WO 03/028661 PCT/US02/31726
[145] Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987)
Supplement 30.
[146] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.
22
