Note: Descriptions are shown in the official language in which they were submitted.
CA 02765058 2012-01-13
IMMUNOGENIC COMPOSITIONS BASED ON
MICROPARTICLES COMPRISING ADSORBED TOXOID AND
A POLYSACCHARIDE-CONTAINING ANTIGEN
This application is a divisional application of Canadian Patent Application
No. 2,528,007 filed on June 2, 2004.
Field of the Invention
[0002] The present invention relates to immunogenic pharmaceutical
compositions,
particularly vaccine compositions.
Background =
[0003] The emergence of subunit vaccines, including polypeptide,
polysaccharide,
conjugate, and DNA vaccines, has intensified the need for safe and effective
adjuvant-
containing compositions.
_ - [0004] Currently, the most commonly used adjuvants in the
United States are alum
adjuvants (i.e., aluminum salts such as aluminum hydroxide and aluminum
phosphate).
Only alum is currently approved for human use by the U.S. Department of Health
and
Human Services, Food and Drug Administration (FDA). For example, various
diphtheria-tetanus vaccines are available in which diphtheria toxoids and
tetanus toxoids
are adsorbed to aluminum salts. Although aluminum adjuvants have a
demonstrated
safety profile of many years, these adjuvants are nonetheless occasionally
associated with
local reactions. For example, post-vaccination granulomas are a well-known
reaction
associated with aluminum-adsorbed vaccines.
[0005] Particulate carriers have been used with adsorbed or entrapped antigens
in
attempts to elicit adequate immune responses. Such carriers typically present
multiple
copies of a selected recombinant protein antigen to the immune system and
promote
1
CA 02765058 2012-01-13
trapping and retention of antigens in local lymph nodes. The particles can be
phagocytosed by macrophages and can enhance antigen presentation through
cytokine
release.
[0006] For example, commonly owned International patent application WO
98/33487 and
co-pending U.S. Patent Application Serial No. 09/015,652, filed January 29,
1998,
describe the use of antigen-adsorbed and antigen-encapsulated microparticles
to stimulate
immunological responses, including cell-mediated immunological responses, as
well as
methods of making the microparticles. Polymers used to form the microparticles
include
poly(lactide) and poly(lactide-co-glycolide), also referred to herein as
"PLG".
[0007] Commonly owned International patent application WO 00/06123 and co-
pending
U.S. Patent Application Serial No. 09/715,902 disclose methods of making
microparticles
having adsorbed macromolecules, including DNA, polypeptides, antigens and
adjuvants.
The microparticles comprise, for example, a polymer such as a poly(alpha-
hydroxy acid)
(e.g., PLG), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester,
a
polyanhydride, and the like and are formed using, for example, cationic,
anionic or
nonionic detergents. Microparticles containing anionic detergents, such as PLG
microparticles with sodium dodecyl sulfate (SDS), are proposed for the use of
positively
charged macromolecules, such as polypeptides. Microparticles containing
cationic
detergents, such as PLG microparticles with CTAB (cetyltrimethylammonium
bromide),
are proposed for the use of negatively charged macromolecules, such as DNA.
The use
of such microparticles to stimulate immunological responses, including cell-
mediated
immunological responses, is also disclosed.
=
Summary of the Invention
[0008] The present invention relates to immunogenic compositions comprising
biodegradable polymer microparticles having toxoid and polysaccharide-
containing
antigens adsorbed thereto.
[0009] According to a first aspect of the invention, an immunogenic
composition is
provided which comprises: (a) polymer microparticles comprising a
biodegradable
polymer, for example, a polymer selected from a poly(a-hydroxy acid), a
polyhydroxy
butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, and a
polycyanoacrylate; (b) an antigen adsorbed to the microparticles selected from
(i) a toxoid
2
CA 02765058 2012-01-13
antigen, such as a tetanus toxoid, a diphtheria toxoid, or a combination
thereof, and/or (ii)
a polysaccharide containing antigen, such as a Hib polysaccharide antigen, a
Hib
conjugate antigen comprising polysaccharide and polypeptide regions, a
meningococcal
polysaccharide antigen, a meningococcal conjugate antigen comprising
polysaccharide
and polypeptide regions, a pneumococcal polysaccharide antigen, a pneumococcal
conjugate antigen comprising polysaccharide and polypeptide regions, or a
combination
thereof; and (c) a pharmaceutically acceptable excipient. Typically,
microparticles are
prepared via any of a variety of techniques, after which the antigen is
adsorbed to the
microparticles.
[0010] In many embodiments, the microparticles are formed from a poly(a-
hydroxy
acid), such as a poly(lactide) ("PLA"), a copolymer of lactide and glycolide,
such as a
poly(D,L-lactide-co-glycolide) ("PLG"), or a copolymer of D,L-lactide and
caprolactone.
Poly(D,L-lactide-co-glycolide) polymers include those having a
lactide:glycolide molar
ratio ranging, for example, from 20:80 to 80:20, from 25:75 to 75:25, from
40:60 to
60:40, or from 55:45 to 45:55, and having a molecular weight ranging, for
example, from
5,000 to 200,00 Daltons, from 10,000 to 100,000 Daltons, from 20,000 to 70,000
Daltons,
or from 40,000 to 50,000 Daltons.
[0011] In many embodiments, the immunogenic compositions will comprise
antigens in
addition to toxoid and/or polysaccharide-containing antigens. These additional
antigens
may independently be, for example: (a) adsorbed to the surface of the
microparticles, (b)
entrapped within the microparticles, (c) in solution or suspension, (d)
adsorbed to separate
populations of microparticles, and/or (e) entrapped within separate
populations of
microparticles.
[0012] Antigens can be, for example, killed or attenuated pathogens (e.g.,
bacteria,
viruses, fungi or parasites) or cells (e.g., tumor cells), polypeptide
containing antigens,
polysaccharide containing antigens, toxoids, or polynucleotide containing
antigens.
[0013] Examples of polynucleotide-containing antigens include, for example,
(a) nucleic
acid sequences that directly encode polypeptide-containing antigens (e.g., an
mRNA
molecule) and (b) vector constructs that indirectly encode polypeptide-
containing
antigens, for example, vector constructs that express heterologous nucleic
acid sequences,
which in turn encode polypeptide-containing antigens (e.g., DNA vector
constructs and
3
CA 02765058 2012-01-13
RNA vector constructs). The encoded polypeptide-containing antigens can be,
for
example, tumor antigens and/or antigens derived from pathogenic organisms.
[0014) Similarly, polypeptide containing antigens and polysaccharide
containing antigens
can be, for example, tumor antigens and/or pathogenic organism antigens. Thus,
in some
embodiments, these antigens are derived from tumors. In other embodiments, the
antigens are derived from viruses, for example, hepatitis virus, herpes
simplex, human
immunodeficiency virus, varicella virus, polio, measles, mumps, rubella,
cytomegalovirus
and influenza virus. In other embodiments, the antigens are derived from
bacteria such
as, for example, diphtheria, tetanus, pertussis, Neisseria meningitidis,
Haemophilus
influenza (e.g., Haemophilus influenza type b), streptococcus, Neisseria
gonorrhoeae,
Helicobacter pylori, and cholera. In still other embodiments, the antigens are
derived
from fungi, or from parasites such as, for example, a malaria parasite.
[0015] Specific examples of antigens include various combinations of the
following: (a)
pertussis antigens (e.g., whole-cell and acellular pertussis antigens), (b)
Haemophilus
influenzae type b (Hib) antigens (e.g., Hib polysaccharide and Hib conjugate
antigens),
(c) hepatitis antigens (e.g., hepatitis A virus antigens, hepatitis C virus
antigens, hepatitis
D virus antigens, hepatitis E virus antigens, hepatitis G virus antigens, and
combinations
thereof, e.g., hepatitis A-B); (d) polio antigens (e.g., killed and live
attenuated virus
antigens, typically trivalent inactivated polio antigens); (e) meningococcal
(Neisseria
meningitidis) antigens, including polysaccharide and conjugate antigens (e.g.,
meningitis
A, meningitis B, meningitis C, meningitis W, meningitis Y and combinations,
such as
meningitis A-C, meningitis A-B-C, meningitis A-C-W-Y and meningitis A-B-C-W-
Y);
(f) pneumococcal (Streptococcus pneumoniae) antigens (e.g., polysaccharide and
conjugate antigens); (g) varicella zoster virus (chickenpox) antigens (e.g.,
lyophilized,
live attenuated virus antigens), (h) measles virus antigens (e.g., live
attenuated virus
antigens), (i) mumps virus antigens (e.g., live, attenuated virus antigens),
(j) rubella virus
antigen (e.g., live, attenuated virus antigens).
[0016] The immunogenic compositions of the present invention can also comprise
various supplementary immunological adjuvants. As with the additional antigens
above,
these supplementary immunological adjuvants may independently be, for example:
(a)
adsorbed to the surface of the microparticles, (b) entrapped within the
microparticles, (c)
4
CA 02765058 2012-01-13
in solution/suspension, (d) adsorbed to separate populations of
microparticles, and/or (e)
entrapped within separate populations of microparticles.
[0017] Examples of supplementary immunological adjuvants include (a)
immunostimulating oligonucleotides such as CpG oligonucleotides, (b) double-
stranded
RNA, (c) E. coli heat-labile toxins, (d) liposaccharide phosphate compounds
(e.g.,
monophosphorylipid A and derivatives) and liposaccharide phosphate mimetics,
and (e)
submicron emulsions comprising a metabolizable oil, such as squalene, and an
emulsifying agent, such as one or more sorbitan derivatives (e.g., MF59).
[0018] Still other supplementary components can be included within the various
compositions of the present invention, including pharmaceuticals, hormones,
enzymes,
transcription or translation mediators, metabolic pathway intermediates,
immunomodulators, and combinations thereof.
[0019] Further embodiments of the invention are directed to methods of
delivering
antigens to a host animal, which comprises administering to the host animal
any of the
immunogenic compositions described herein. The host animal is preferably a
vertebrate
animal, more preferably a mammal, and even more preferably a human.
[0020] The present invention is also directed to methods of stimulating an
immune
response in a host animal, comprising administering to the animal any of the
immunogenic compositions described herein in an amount effective to induce the
immune
response.
[0021] The present invention is further directed to methods of immunizing a
host animal
against a tumor or a pathogenic organism comprising administering to the
animal any of
the immunogenic compositions described herein in an amount effective to induce
a
protective response.
[0022] Delivery of the immunogenic compositions of the invention may be
performed by
any known method, including direct injection (e.g., subcutaneously,
intraperitoneally,
intravenously or intramuscularly).
[0023] Hence, according to some embodiments of the present invention,
compositions
and methods are provided which treat, including prophylactically and/or
therapeutically
immunize, a host animal against viral, bacterial, fungal, mycoplasma, or
protozoan
infections, as well as against tumors. The methods of the present invention
are useful for
CA 02765058 2014-11-28
conferring prophylactic and/or therapeutic immunity to a host animal,
preferably a
human.
[0024] Other embodiments of the present invention are directed to methods for
producing the above compositions. For example, the above polymer
microparticles can
be produced by a method that comprises: (a) forming a water-in-oil-in-water
emulsion
comprising water, organic solvent, biodegradable polymer; (b) removing the
organic
solvent from the emulsion, to form the polymer microparticles; and (c)
adsorbing a
toxoid antigen, such as a tetanus or diphtheria toxoid, a polysaccharide
containing
antigen, such as a Hib, meningococcal or pneumococcal polysaccharide or
conjugate
antigen, or a combination thereof, to the microparticles. In many embodiments
the
emulsion will further comprise a surfactant, for example, an anionic
surfactant.
[0025] One advantage of the immunogenic compositions of the present invention
is the
ability to generate immune responses in a vertebrate subject, including
conventional
antibody responses.
[0025a] In one aspect, there is provided an immunogenic composition
comprising: (a)
polymer microparticles comprising a biodegradable polymer; (b) an antigen
adsorbed to
the microparticles, said antigen selected from one or more of (i) a toxoid
antigen selected
from tetanus toxoid and diphtheria toxoid, and (ii) a polysaccharide-
containing antigen
selected from a meningococcal polysaccharide antigen and a meningococcal
conjugate
antigen comprising polysaccharide and polypeptide regions; and (c) a
pharmaceutically
acceptable excipient, and further comprising an additional polypeptide-
containing
antigen, an additional polysaccharide-containing antigen, an additional
conjugate antigen,
or an additional polynucleotide-containing antigen, adsorbed to the surface of
the
microparticles.
[0025b] In another aspect, there is provided an immunogenic composition
comprising:
(a) polymer microparticles comprising a biodegradable polymer; (b) an antigen
adsorbed
to the microparticles, said antigen selected from one or more of (i) a toxoid
antigen
selected from tetanus toxoid and diphtheria toxoid, and (ii) a polysaccharide-
containing
antigen selected from a meningococcal polysaccharide antigen and a
meningococcal
conjugate antigen comprising polysaccharide and polypeptide regions; and (c) a
pharmaceutically acceptable excipient, and further comprising an additional
polypeptide-
containing antigen, an additional polysaccharide-containing antigen, an
additional
6
CA 02765058 2014-11-28
conjugate antigen, or an additional polynucleotide-containing antigen,
entrapped within
the microparticles.
[0026] These and various other embodiments, aspects and advantages of the
present
invention will become readily apparent to those of ordinary skill in the art
in view of the
disclosure herein and the appended claims.
Brief Description of the Figures
[0027] Fig. 1 is the Recommended Childhood and Adolescent Immunization
Schedule¨
United States 2003, from the U.S. Department of Health and Human Services,
Centers
for Disease Control and Prevention, National Immunization Program.
[0028] Fig. 2 is a bar graph illustrating % adsorption and % release for
tetanus toxoid and
diphtheria toxoid from PLG particles in Histidine buffer.
[0029] Fig. 3 is a bar graph illustrating % adsorption and % release for Men-X
Crm
conjugate from PLG particles.
Detailed Description of the Invention
[0030] The practice of the present invention will employ, unless otherwise
indicated,
conventional methods of chemistry, polymer chemistry, biochemistry, molecular
biology,
immunology and pharmacology, within the skill of the art. Such techniques are
explained
fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th
Edition
6a
CA 02765058 2012-01-13
(Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology
(S.
Colowick and N. Kaplan, eds., Academic Press, Inc.); Handbook of Experimental
Immunology, V ols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell
Scientific
= Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual
(2nd Edition,
1989); Handbook of Surface and Colloidal Chemistry (Birth, K.S., eci, CRC
Press, 1997)
and Seymour/Carraher=s Polymer Chemistry (4th edition, Marcel Dekker Inc.,
1996).
[0032] As used in this specification and any appended claims, the singular
forms "a,"
"an" and "the" include plural references unless the content clearly dictates
otherwise.
Thus, for example, the term "microparticle" refers to one or more
microparticles, and the
like.
[0033] Unless the context indicates otherwise, all percentages and ratios
herein are given
on a weight basis.
A. Definitions
[0034] In describing the present invention, the following terms will be
employed, and are
intended to be defined as indicated below.
[0035] The term "microparticle" as used herein, refers to a particle of about
10 nm to
about 150 pm in diameter, more typically about 200 run to about 30 gm in
diameter, and
even more typically about 500 nm to about 10-20 pm in diameter. The
microparticles of
the present invention may aggregate into larger masses under some
circumstances. The
microparticle will generally be of a diameter that permits parenteral or
mucosal
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 "particle"
may also
be used to denote a microparticle as defmed herein.
[0036] Polymer microparticles for use herein are typically formed from
materials that are
sterilizable, substantially non-toxic, and biodegradable. Such materials
include poly(a-
hydroxy acids), polyhydroxybutyric acids, polycaprolactones, polyorthoesters,
polyanhydrides, and polycyanoacrylates (e.g., polyalkylcyanoacrylate or
"PACA"). More
typically, microparticles for use with the present invention are polymer
microparticles
7
CA 02765058 2012-01-13
derived from poly(a-hydroxy acids), for example, from a poly(lactide) ("PLA")
or a
copolymer of lactide and glycolide, such as a poly(D,L-lactide-co-glycolide)
("PLG"), or
a copolymer of D,L-lactide and caprolactone. The polymer microparticles may be
derived from any of various polymeric starting materials which have a variety
of
molecular weights and, in the case of the copolymers such as PLG, a variety of
monomer
(e.g., lactide:glycolide) ratios, the selection of which will be largely a
matter of choice,
depending in part on the coadministered species. These parameters are
discussed further
below.
[0037] The term "surfactant" as used herein includes detergents, dispersing
agents,
suspending agents, and emulsion stabilizers. Cationic surfactants include, but
are not
limited to, cetyltrimethylammonium bromide or "CTAB" (e.g., cetrimide),
benzalkonium
chloride, DDA (dimethyl dioctodecyl ammonium bromide), DOTAP (dioleoy1-3-
trimethylammonium-propane), and the like. Anionic surfactants include, but are
not
limited to, SDS (sodium dodecyl sulfate), SLS (sodium lauryl sulfate), DSS
=
(disulfosuccinate), sulphated fatty alcohols, and the like. Nonionic
surfactants include,
but are not limited to, PVA, povidone (also known as polyvinylpyrrolidone or
PVP),
sorbitan esters, polysorbates, polyoxyethylated glycol monoethers,
polyoxyethylated alkyl
phenols, poloxamers, and the like.
[0038] The term "submicron emulsion" as used herein refers to an oil-in-water
emulsion
comprising oil droplets, substantially all of which range in size up to 1000
nm, for
example, from 10 nm to 1000 nm.
[0039] The term "pharmaceutical" refers to biologically active compounds such
as
antibiotics, antiviral agents, growth factors, hormones, antigens and the
like.
[0040] The term "adjuvant" refers to any substance that assists or modifies
the action of a
pharmaceutical, including but not limited to immunological adjuvants, which
increase or
diversify the immune response to an antigen. Hence, immunological adjuvants
are
compounds that are capable of potentiating an immune response to antigens.
[0041] A "polynucleotide" is a nucleic acid polymer. A polynucleotide can
include as
little as 5, 6, 7 or 8 nucleotides. Furthermore, a "polynucleotide" can
include both
double- and single-stranded sequences and refers to, but is not limited to,
cDNA from
viral, procaryotic or eukaryotic mRNA, genomic RNA and DNA sequences from
viral
(e.g. RNA and DNA viruses and retroviruses) or procaryotic DNA, and synthetic
DNA
8
CA 02765058 2012-01-13
sequences. The term also captures sequences that include any of the known base
analogs
of DNA and RNA. The term further includes modifications, such as deletions,
additions
and substitutions (generally conservative in nature), to a native sequence,
for example;
where the nucleic acid molecule encodes an antigenic protein. These
modifications may
be deliberate, as through site-directed mutagenesis, or may be accidental,
such as through
mutations of hosts that produce antigens.
[0042] As used herein, the phrase "nucleic acid" refers to DNA, RNA, or
chimeras
formed therefrom.
[0043] A "polynucleotide-containing species" is a molecule, at least a portion
of which is
a polynucleotide. Examples include RNA vector constructs, DNA vector
constructs and
so forth.
[0044] An "oligosaccharide" refers to a relatively short monosaccharide
polymer, i.e.,
one containing from 2 to 30 monosaccharide units. A "polysaccharide" is a
monosaccharide polymer that is beyond oligosaccharide length (i.e., one
containing more
than 30 monosaccharide units). Moreover, as used herein, the term
"polysaccharide" also
refers to a monosaccharide polymer that contain two or more linked
monosaccharides.
To avoid any ambiguity, the second definition is to be applied at all times,
unless there
are explicit indications to the contrary. The monosaccharides are typically
linked by
glycosidic linkages. Both full-length, naturally occurring polysaccharides and
fragments
thereof are encompassed by the definition. The terms also include
modifications, such as
deletions, additions and substitutions to a native polysaccharide sequence,
for example,
such that the polysaccharide maintains the ability to elicit an immunological
response on
a subject to which the polysaccharide is administered.
[0045] A "monosaccharide" is a polyhydric alcohol (i.e., an alcohol that
further
comprises either an aldehyde group (in which case the monosaccharide is an
aldose) or a
keto group (in which case the monosaccharide is a ketose). Monosaccharides
typically
contain from 3-10 carbons. Moreover, monosaccharides typically have the
empirical
formula (CH20)õ where n is an integer of three or greater, typically 3-10.
Examples of 3-
6 carbon aldoses include glyceraldehyde, erythrose, threose, ribose, 2-
deoxyribose,
arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose,
galactose, and
talose. Examples of 3-6 carbon ketoses include dihydroxyacetone, erythrulose,
ribulose,
9
CA 02765058 2012-01-13
xylulose, psicose, fructose, sorbose, and tagatose. Naturally occurring
monosaccharides
are normally found in the D-isomer form, as opposed to the L-forrn.
[0046] As used herein the term "saccharide" encompasses monosaccharides,
oligosaccharides and polysaccharides. A "saccharide-containing species" is a
molecule,
at least a portion of which is a saccharide. Examples include saccharide
antigens,
antigens comprising saccharides conjugated to carrier peptides, and so forth.
[0047] The terms "polypeptide" and "protein" refer to a polymer of amino acid
residues
and are not limited to a minimum length of the product. Thus, peptides,
oligopeptides,
dimers, multimers, and the like, are included within the definition. Both full-
length
proteins and fragments thereof are encompassed by the definition. The terms
also include
modifications, such as deletions, additions and substitutions (generally
conservative in
nature), to a native sequence, for example, such that the protein maintains
the ability to
elicit an immunological response or have a therapeutic effect on a subject to
which the
protein is administered.
[0048] A "polypeptide-containing species" is a molecule, at least a portion of
which is a
polypeptide. Examples include polypeptides, proteins including glycoproteins,
saccharide antigens conjugated to carrier proteins, and so forth.
[0049] By "antigen" is meant a molecule that contains one or more epitopes
capable of
stimulating a host's immune system to make a cellular antigen-specific immune
response
when the antigen is presented, or a humoral antibody response. An antigen may
be
capable of eliciting a cellular and/or humoral response by itself or when
present in
combination with another molecule.
[0050] An "epitope" is that portion of an antigenic molecule or antigenic
complex that =
determines its immunological specificity. An epitope is within the scope of
the present
definition of antigen. Commonly, an epitope is a polypeptide or polysaccharide
in a
naturally occurring antigen. In artificial antigens, it can be a low molecular
weight
substance such as an arsanilic acid derivative. An epitope will react
specifically in vivo or
in vitro with, for example, homologous antibodies or T lymphocytes.
Alternative
descriptors are antigenic determinant, antigenic structural grouping and
haptenic
grouping.
[0051] A polypetide epitope can include, for example, between about 5-15 amino
acids.
CA 02765058 2012-01-13
Epitopes of a given antigen can be identified using any number of epitope
mapping
techniques, well known in the art. See, e.g., Epitope Mapping Protocols in
Methods in
Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa,
New
Jersey. For example, linear epitopes may be determined by, for example,
concurrently
synthesizing large numbers of peptides on solid supports, the peptides
corresponding to
portions of the protein molecule, and reacting the peptides with antibodies
while the
peptides are still attached to the supports. Such techniques are known in the
art and
described in, e.g., U.S. Patent No. 4,708,871; Geysen etal. (1984) Proc. Natl.
Acad. Sci.
USA 81:3998-4002; Geysen etal. (1986) Molec. Immunol. 23:709-715. Similarly,
conformational epitopes are readily identified by determining spatial
conformation of
amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear
magnetic
resonance. See, e.g., Epitope Mapping Protocols, supra.
[0052] The term "antigen" as used herein denotes both subunit antigens, i.e.,
antigens
which are separate and discrete from a whole organism with which the antigen
is
associated in nature, as well as killed, attenuated or inactivated bacteria,
viruses, parasites
or other pathogens or tumor cells. Antibodies such as anti-idiotype
antibodies, or
fragments thereof and synthetic peptide mimotopes, which can mimic an antigen
or
antigenic determinant, are also captured under the definition of antigen as
used herein.
[0053] Similarly, an oligonucleotide or polynucleotide (oligonucleotides are
polynucleotides of relatively low molecular weight, typically containing from
2 to 100
nucleotides) that expresses an immunogenic protein, or antigenic determinant
in vivo,
such as in nucleic acid immunization applications, is also included in the
definition of
* antigen herein.
[0054] -Furthermore, for purposes of the present invention, an "antigen"
refers to a
protein, which includes modifications, such as deletions, additions and
substitutions
(generally conservative in nature), to the native sequence, as long as the
protein maintains
the ability to elicit an immunological response. These modifications may be
deliberate,
as through site-directed mutagenesis, or may be accidental, such as through
mutations of
hosts which produce the antigens.
[0055] An "immunological response" to an antigen or composition is the
development in
a subject of a humoral and/or a cellular immune response to molecules present
in the
composition of interest. For purposes of the present invention, a "humoral
immune
11
CA 02765058 2012-01-13
response" refers to an immune response mediated by antibody molecules, while a
"cellular immune response" is one mediated by T-lymphocytes and/or other white
blood
cells. One important aspect of cellular immunity involves an antigen-specific
response by
cytolytic T-cells ("CTLs"). CTLs have specificity for peptide antigens that
are presented
in association with proteins encoded by the major histocompatibility complex
(MHC) and
expressed on the surfaces of cells. CTLs help induce and promote the
intracellular
destruction of intracellular microbes, or the lysis of cells infected with
such microbes.
Another aspect of cellular immunity involves an antigen-specific response by
helper T-
cells. Helper T-cells act to help stimulate the function, and focus the
activity of;
nonspecific effector cells against cells displaying peptide antigens in
association with
MHC molecules on their surface. A "cellular immune response" also refers to
the
production of cytolcines, chemolcines and other such molecules produced by
activated T-
cells and/or other white blood cells, including those derived from CD4+ and
CD8+ T-
cells. A composition such as an immunogenic composition or vaccine that
elicits a
cellular immune response may serve to sensitize a vertebrate subject by the
presentation
of antigen in association with MHC molecules at the cell surface. The cell-
mediated
immune response is directed at, or near, cells presenting antigen at their
surface. In
addition, antigen-specific T-lymphocytes can be generated to allow for the
future
protection of an immunized host. The ability of a particular antigen or
composition to
stimulate a cell-mediated immunological response may be determined by a number
of
assays, such as by lymphoproliferation (lymphocyte activation) assays, cm
cytotoxic
cell assays, by assaying for T-lymphocytes specific for the antigen in a
sensitized subject,
or by measurement of cytokine production by T cells in response to
restimulation with
antigen. Such assays are well known in the art. See, e.g., Erickson et al., J.
Immunol.
(1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994) 2:2369-2376. The
antigen of
interest may also elicit an antibody-mediated immune response. Hence, an
immunological response may include one or more of the following effects: the
production
of antibodies by B-cells; and/or the activation of suppressor T-cells and/or
78 T-cells
directed specifically to an antigen or antigens present in the composition or
vaccine of
interest. These responses may serve to neutralize infectivity, and/or mediate
antibody-
complement, or antibody dependent cell cytotoxicity (ADCC) to provide
protection :to an
immunized host. Such responses can be determined using standard immunoassays
and
12
CA 02765058 2012-01-13
neutralization assays, well known in the art, for instance, radioimmunoassays
and
ELISAs.
[0056] The immunogenic compositions of the present invention display "enhanced
immunogenicity" when they possess a greater capacity to elicit an immune
response than
the immune response elicited by an equivalent amount of the antigen in a
differing
composition. Thus, a composition may display "enhanced immunogenicity," for
example, because the composition generates a stronger immune response, or
because a
lower dose of antigen is necessary to achieve an immune response in the
subject to which
it is administered. Such enhanced immunogenicity can be determined, for
example, by
administering the compositions of the invention, and antigen controls, to
animals and
comparing assay results of the two.
[0057] As used herein, "treatment" (including variations thereof, for example,
"treat" or
"treated") refers to any of (i) the prevention of a pathogen or disorder in
question (e.g.
cancer or a pathogenic infection, as in a traditional vaccine), (ii) the
reduction or
elimination of symptoms, and (iii) the substantial or complete elimination of
the pathogen
or disorder in question. Treatment may be effected prophylactically (prior to
arrival of
the pathogen or disorder in question) or therapeutically (following arrival of
the same).
[0058] The terms "effective amount" or "pharmaceutically effective amount" of
an
immunogenic composition of the present invention refer herein to a sufficient
amount of
the immunogenic composition to treat a condition of interest. The exact amount
required
will vary from subject to subject, depending, for example, on the species,
age, and general
condition of the subject; the severity of the condition being treated; the
particular antigen
of interest; in the case of an immunological response, the capacity of the
subject's
immune system to synthesize antibodies, for example, and the degree of
protection
desired; and the mode of administration, among other factors. An appropriate
"effective"
amount in any individual case may be determined by one of ordinary skill in
the art. Thus,
a "therapeutically effective amount" will typically fall in a relatively broad
range that can
be determined through routine trials.
[0059] By "vertebrate subject" or "vertebrate animal" is meant any member of
the
subphylum cordata, including, without limitation, mammals such as cattle,
sheep, pigs,
goats, horses, and humans; domestic animals such as dogs and cats; and birds,
including
domestic, wild and game birds such as cocks and hens including chickens,
turkeys and
13
CA 02765058 2012-01-13
=
other gallinaceous birds. The term does not denote a particular age. Thus,
both adult and
newborn animals are covered.
[0060] By "pharmaceutically acceptable" or "pharmacologically acceptable" is
meant a
material which is not biologically or otherwise undesirable, i.e., the
material may be
administered to an individual without causing any excessively undesirable
biological
effects in the individual or interacting in an excessively deleterious manner
with any of
the components of the composition in which it is contained.
[0061] The term "excipient" refers to any essentially accessory substance that
may be
present in the finished dosage form. For example, the term "excipient"
includes vehicles,
binders, disintegrants, fillers (diluents), lubricants, glidants (flow
enhancers),
compression aids, colors, sweeteners, preservatives, suspending/dispersing
agents, film
formers/coatings, flavors and printing inks.
[0062] By "physiological pH" or a "pH in the physiological range" is meant a
pH in the
range of approximately 7.2 to 8.0 inclusive, more typically in the range of
approximately
7.2 to 7.6 inclusive.
[0063] As used herein, the phrase "vector construct" generally refers to any
assembly that
is capable of directing the expression of a nucleic acid sequence(s) or
gene(s) of interest.
A vector construct typically includes transcriptional promoter/enhancer or
locus defining
element(s), or other elements which control gene expression by other means
such as
alternate splicing, nuclear RNA export, post-translational modification of
messenger, or
post-transcriptional modification of protein. In addition, the vector
construct typically
includes a sequence which, when transcribed, is operably linked to the
sequence(s) or
gene(s) of interest and acts as a translation initiation sequence. The vector
construct may
also optionally include a signal that directs polyadenylation, a selectable
marker, as well
as one or more restriction sites and a translation termination sequence. In
addition, if the
vector construct is placed into a retrovirus, the vector construct may include
a packaging
signal, long terminal repeats (Lilts), and positive and negative strand primer
binding
sites appropriate to the retrovirus used (if these are not already present).
[0064] A "DNA vector construct" refers to a DNA molecule that is capable of
directing
the expression of a nucleic acid sequence(s) or gene(s) of interest.
[0065] One specific type of DNA vector construct is a plasmid, which is a
circular
14
CA 02765058 2012-01-13
episomal DNA molecule capable of autonomous replication within a host cell.
Typically,
a plasmid is a circular double stranded DNA loop into which additional DNA
segments
can be ligated. pCMV is one specific plasmid that is well known in the art. A
preferred
pCMV vector is one which contains the immediate-early enhancer/promoter of CMV
and
a bovine growth hormone terminator. It is described in detail in Chapman, B.
S., et al.
1991. "Effect of intron A from human cytomegalovirus (Towne) immediate-early
gene on
heterologous expression in mammalian cells." Nucleic Acids Res. 19:3979-86.
[0066] Other DNA vector constructs are known, which are based on RNA viruses.
These
DNA vector constructs typically comprise a promoter that functions in a
eukaryotic cell,
5' of a cDNA sequence for which the transcription product is an RNA vector
construct
(e.g., an alphavirus RNA vector replicon), and a 3' termination region. The
RNA vector
construct preferably comprises an RNA genome from a picomavirus, togavims,
flavivirus, coronavirus, paramyxovirus, yellow fever virus, or alphavirus
(e.g., Sindbis
virus, Semliki Forest virus, Venezuelan equine encephalitis virus, or Ross
River virus),
which has been modified by the replacement of one or more structural protein
genes with
a selected heterologous nucleic acid sequence encoding a product of interest.
The RNA
vector constructs can be obtained by transcription in vitro from a DNA
template. Specific
examples include Sindbis-virus-based plasmids (pSI/1) such as pSlNCP ,
described, for
example, in U.S. Patents 5,814,482 and 6,015,686, as well as in International
Patent
Applications WO 97/38087, WO 99/18226 and commonly owned WO 02/26209. The
construction of such vectors, in general, is described in U.S. Patents
5,814,482 and
6,015,686.
[0067] Other examples of vector constructs include RNA vector constructs
(e.g.,
alphavirus vector constructs) and the like. As used herein, "RNA vector
construct",
"RNA vector replicon" and "replicon" refer to an RNA molecule that is capable
of
directing its own amplification or self-replication in vivo, typically within
a target cell.
The RNA vector construct is used directly, without the requirement for
introduction of
DNA into a cell and transport to the nucleus where transcription would occur.
By using
the RNA vector for direct delivery into the cytoplasm of the host cell,
autonomous
replication and translation of the heterologous nucleic acid sequence occurs
efficiently. .
CA 02765058 2012-01-13
B. General Methods
1. Microparticle Compositions
[0068] Useful polymers for forming the immunogenic microparticle compositions
described herein include homopolymers, copolymers and polymer blends derived
from
the following: polyhydroxybutyric acid (also known as polyhydroxybutyrate);
polyhydroxy valeric acid (also known as polyhydroxyvalerate); polyglycolic
acid (PGA)
(also known as polyglycolide); polylactic acid (PLA) (also known as
polylactide);
polydioxanone; polycaprolactone; polyorthoester; and polyanhydride. More
typical are
poly(a-hydroxy acids), such as poly(L-lactide), poly(D,L-lactide) (both
referred to as
APLA" herein), poly(hydoxybutyrates), copolymers of lactide and glycolide,
such as
poly(D,L-lactide-co-glycolides) (designated as "PLG" herein), or copolymers of
D,L-
lactide and caprolactone.
[0069] The above polymers are available in a variety of molecular weights, and
the
appropriate molecular weight for a given use is readily determined by one of
skill in the
art. Thus, for example, a suitable molecular weight for PLA may be on the
order of about
2000 to 5000. A suitable molecular weight for PLG may range from about 10,000
to
about 200,000, typically about 15,000 to about 150,000.
[0070] Where copolymers are used, copolymers with a variety of monomer ratios
may be
available. For example, where PLG is used to form the microparticles, a
variety of
lactide:glycolide molar ratios will find use herein, and the ratio is largely
a matter of
choice, depending in part on any coadministered adsorbed and/or entrapped
species 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. Mixtures of microparticles with varying lactide:glycolide
ratios may
also find use herein in order to achieve desired release kinetics. Degradation
rate of the
microparticles of the present invention can also be controlled by such factors
as polymer
molecular weight and polymer crystallinity.
[0071] PLG copolymers with varying lactide:glycolide ratios and molecular
weights are
readily available commercially from a number of sources including from
Boehringer
Ingelheim, Germany and Birmingham Polymers, Inc., Birmingham, AL. Some
exemplary PLG copolymers include: (a) RG 502, a PLO having a 50:50
lactide/glycolide
16
CA 02765058 2012-01-13
molar ratio and a molecular weight of 12,000 Da; (b) RG 503, a PLG having a
50:50
lactide/glycolide molar ratio and a molecular weight of 34,000 Da; (c) RG 504,
a PLG
having a 50:50 lactide/glycolide molar ratio and a molecular weight of 48,000
Da, (d) RG
752, a PLG having a 75:25 lactide/glycolide molar ratio and a molecular weight
of 22,000
Da; and (e) RG 755, a PLG having a 75:25 lactide/glycolide molar ratio and a
molecular
weight of 68,000 Da. PLG polymers can also be synthesized by simple
polycondensation of the lactic acid component using techniques well known in
the art,
such as described in Tabata et al., J. Biomed Mater. Res. (1988) 22:837-858.
[0072] Where used, poly(D,L-lactide-co-glycolide) polymers are typically those
having a
molar lactide/glycolide molar ratio ranging from 20:80 to 80:20, more
typically 40:60 to
60:40, and having a molecular weight ranging from 10,000 to 100,000 Daltons,
more
typically from 20,000 Daltons to 70,000 Daltons.
[0073] Microparticles are prepared using any of several methods well known in
the art.
For example, in some embodiments, double emulsion/solvent evaporation
techniques,
such as those described in U.S. Patent No. 3,523,907 and Ogawa et al., Chem.
Pharm.
Bull. (1988) 36:1095-1103, can be used herein to make the microparticles.
These
techniques involve the formation of a primary emulsion consisting of droplets
of polymer
solution, which is subsequently mixed with a continuous aqueous phase
containing a
particle stabilizer/surfactant.
[0074] In other embodiments, microparticles can also be formed using spray-
drying and
coacervation as described in, e.g., Thomasin et al., J Controlled Release
(1996) 41:131;
U.S. Patent No. 2,800,457; Masters, K. (1976) Spray Drying 2nd Ed. Wiley, New
York; =
air-suspension coating techniques, such as pan coating and Wurster coating, as
described
by Hall et al., (1980) The AWurster Process@ in Controlled Release
Technologies:
Methods, Theory, and Applications (A.F. Kydonieus, ed.), Vol. 2, pp. 133-154
CRC
Press, Boca Raton, Florida and Deasy, P.B., Crit. Rev. Ther. Drug Carrier
Syst. (1988)
S(2):99-139; and ionic gelation as described by, e.g., Lim et al., Science
(1980) 210:908-
910.
[0075] In preferred embodiments, a water-in-oil-in-water (w/o/w) solvent
evaporation
system can be used to form the microparticles, along the lines described by
O'Hagan et
al., Vaccine (1993) 11:965-969, PCT/US99/17308 (WO 00/06123) to O'Hagan et al.
and
Jeffery et al., Pharm. Res. (1993) 10:362.
17
CA 02765058 2012-01-13
[0076] In general, a polymer of interest such as PLG is dissolved in an
organic solvent,
such as ethyl acetate, dimethylchloride (also called methylene chloride and
dichloromethane), acetonitrile, acetone, chloroform, and the like. The polymer
will
typically be provided in about a 1-30%, more typically about a 2-15%, even
more
typically about a 3-10% and most typically, about a 4-8% solution, in organic
solvent.
The polymer solution is then combined with a first volume of aqueous solution
and
emulsified to form an o/w emulsion. The aqueous solution can be, for example,
deionized water, normal saline, a buffered solution, for example, phosphate-
buffered
saline (PBS) or a sodium citrate/ethylenediaminetetraacetic acid (sodium
citrate/ETDA)
buffer solution. The latter solutions can (a) provide a tonicity, i.e.,
osmolality, that is
essentially the same as normal physiological fluids and (b) maintain a pH
compatible with
normal physiological conditions. Alternatively, the tonicity and/or pH
characteristics of
the compositions of the present invention can be adjusted after microparticle
formation
and prior to administration. Preferably, the volume ratio of polymer solution
to aqueous
solution ranges from about 5:1 to about 20:1, more preferably about 10:1.
Emulsification
is conducted using any equipment appropriate for this task, and is typically a
high-shear
device such as, e.g., a homogenizer.
[0077] In some embodiments, one or more additional components are entrapped
within
the microparticles. For example, additional antigen, and/or the supplemental
components
described below can be introduced by adding the same (a) to the polymer
solution, if in
oil-soluble or oil-dispersible form or (b) to the aqueous solution, if in
water-soluble or
water-dispersible form.
[0078] A volume of the o/w emulsion is then combined with a larger second
volume of
an aqueous solution, which typically contains a surfactant. The volume ratio
of aqueous
solution to o/w emulsion typically ranges from about 2:1 to 10:1, more
typically about
4:1. Examples of surfactants appropriate for the practice of the invention are
listed above.
Those of ordinary skill in the art may readily select surfactants appropriate
for the type of
species to be adsorbed. For example, microparticles manufactured in the
presence of
charged surfactants, such as anionic or cationic surfactants, may yield
microparticles with
a surface having a net negative or a net positive charge, which can adsorb a
wide variety
of molecules. For example, microparticles manufactured with anionic
surfactants, such as
sodium dodecyl sulfate (SDS), e.g., SDS-PLG microparticles, adsorb positively
charged
18
CA 02765058 2012-01-13
species, for example, polypeptide-containing species such as proteins.
Similarly,
microparticles manufactured with cationic surfactants, such as CTAB, e.g.,
PLG/CTAB
microparticles, adsorb negatively charged species, for example, polynucleotide-
containing species such as DNA. Where the species to be adsorbed have regions
of
positive and negative charge, either cationic or anionic or nonionic
surfactants may be
appropriate. Certain species may adsorb more readily to microparticles having
a
combination of surfactants. Moreover, in some instances, it may be desirable
to add
surfactant to the above organic solution.
[0079] Where a cationic surfactant such as CTAB is used, it is typically
provided in about
a 0.00025-1% solution, more typically about a 0.0025-0.1% solution. Where an
anionic
surfactant such as DSS is used, it is typically provided in about a 0.00001-
.025% solution,
more typically about a 0.0001-0.0025% solution. Where a nonionic surfactant
such as
PVA is used, it is typically provided in about a 2-15% solution, more
typically about a 4-
10% solution. For a cationic surfactant, a weight-to-weight surfactant-to-
polymer ratio
in the range of from about 0.00001:1 to about 0.5:1 is typically used; more
typically from
about 0.001:1 to about 0.1:1, and even more typically from about 0.0025:1 to
about
0.05:1; for an anionic surfactant such as DSS, a weight-to-weight surfactant-
to-polymer
ratio in the range of from about 0.00001:1 to about 0.025:1 is typically used,
more
typically from about 0.0001:1 to about 0.0025:1; for a nonionic surfactant
such as PVA a
weight-to-weight surfactant-to-polymer ratio in the range of from about
0.001:1 to about
0.1:1 is typically used, more typically from about 0.0025:1 to about 0.05:1 is
used.
[0080] This mixture is then homogenized to produce a stable w/o/w double
emulsion.
Each of the above homogenization steps is typically conducted at a room
temperature
(i.e., 25 C) or less, more typically, for example, while cooling within an ice
bath.
[0081] Organic solvents are then evaporated. Following preparation,
microparticles can
be used as is or, for example, lyophilized for future use.
[0082] The formulation parameters can be manipulated to allow the preparation
of small
microparticles on the order of 0.05 pm (50 nm) to larger microparticles 50 um
or even
larger. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee etal.,
J.
Microencap. (1996). For example, reduced agitation typically results in larger
microparticles, as do an increase in internal phase volume and an increase in
polymer
concentration. Small particles are typically produced by increased agitation
as well as
19
CA 02765058 2012-01-13
low aqueous phase volumes, high concentrations of emulsion stabilizers and a
decrease in
polymer concentration.
[0083] Particle size can be determined by, e.g., laser light scattering, using
for example, a
spectrometer incorporating a helium-neon laser. Generally, particle size is
determined at
room temperature and involves multiple analyses of the sample in question
(e.g., 5-10
times) to yield an average value for the particle diameter. Particle size is
also readily
determined using scanning electron microscopy (SEM).
[0084] Upon preparation, a variety of components can be admixed with the
microparticles, including toxoid antigen and/or polysaccharide-containing
antigen, any
additional antigens, and any supplemental components such as those described
below,
and the resulting formulation can be lyophilized prior to use if desired.
Typically, theses
components are added to the microparticles as an aqueous solution or
dispersion. In some
instances, these species will become adsorbed to the surface of the
microparticles (see,
e.g., the Examples below in which toxoid antigens are adsorbed to the
microparticle
surface). The content of the adsorbed species can be determined using standard
techniques.
[0085] Thus, the Polymer microparticles of the present invention may have a
variety of
components adsorbed thereon, as well as having a variety of components
entrapped or
encapsulated within them. For example, one of ordinary skill in the art may
prepare in
accordance with the invention microparticles having adsorbed antigens and/or
immunological adjuvants, in addition to adsorbed toxoid antigen and/or
polysaccharide-
containing antigen. One of ordinary skill in the art may also prepare, in
accordance with
the invention, microparticles having encapsulated components, such as antigens
and/or
immunological adjuvants, in addition to adsorbed toxoid antigen and/or
polysaccharide
containing antigen.
2. Antigens
[0086] The present invention utilizes numerous antigens including toxoid
antigens from
tetanus toxoid, diphtheria toxoid, or both. A toxoid is a toxin that has been
treated so as
to reduce or eliminate its toxicity, while retaining adequate immunogenicity.
Toxoids are
commonly made by treating toxins produced by a bacterium that causes disease
with heat
and/or chemicals. For example, purified diphtheria and tetanus toxoids are
available
CA 02765058 2012-01-13
commercially, which have been prepared by formalin treatment of
Corynebacterium
diphtheriae and Clostridium tetani exotoxins, respectively.
[0087] The present invention will also find use for stimulating an immune
response
against a wide variety of antigens in addition to toxoid antigens.
[0088] For example, antigens from the hepatitis family of viruses, including
hepatitis A
virus (HAV), hepatitis B virus (REV), hepatitis C virus (HCV), the delta
hepatitis virus
(HDV), hepatitis E virus (REV) and hepatitis G virus (HGV), can be
conveniently used in
the techniques described herein. By way of example, the viral genomic sequence
of HCV
is known, as are methods for obtaining the sequence. See, e.g., International
Publication
Nos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodes
several viral proteins, including El (also known as E) and E2 (also known as
E2/NSI) and
an N-terminal nucleocapsid protein (termed "core") (see, Houghton et al.,
Hepatology
(1991) 14:381-388, for a discussion of HCV proteins, including El and E2).
Each of
these proteins, as well as antigenic fragments thereof; will fmd use in the
present
composition and methods.
[00891 Similarly, the sequence for the 5-antigen from HDV is known (see, e.g.,
U.S.
Patent No. 5,378,814) and this antigen can also be conveniently used in the
present
composition and methods. Additionally, antigens derived from HBV, such as the
core
antigen, the surface antigen, sAg, as well as the presurface sequences, pre-S1
and pre-S2
(formerly called pre-S), as well as combinations of the above, such as sAg/pre-
S1,
sAg/pre-S2, sAg/pre-SI/pre-S2, and pre-SI/pre-S2, will find use herein. See,
e.g., AHBV
Vaccines - from the laboratory to license: a case study@ in Mackett, M. and
Williamson,
J.D., Human Vaccines and Vaccination, pp. 159-176, for a discussion of HBV
structure; _
and U.S. Patent Nos. 4,722,840, 5,098,704, 5,324,513;
Bea.mes et al., J. (1995)62:6833-6838, Birnbaum et al., J. Viral.
(1990)fri.1:3319-3330; and Zhou et al., ./. Viral. (1991) 65:5457-5464.
[0090] Antigens from the herpesvirus family, including proteins derived from
herpes
simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycopmteins gB, gD
and
gH; antigens derived from varicella zoster virus (VZV), Epstein-Barr virus
(EBV) and
cytomegalovirus (CMV) including CMV gB and gH; and antigens derived from other
human herpesviruses such as HEIV6 and HHV7 can also be conveniently used in
connection with the present invention. (See, e.g. Chee et al.,
Cytomegaloviruses (J.K.
21
CA 02765058 2012-01-13
McDougall, ed., Springer-Verlag 1990) pp. 125-169, for a review of the protein
coding
content of cytomegalovirus; McGeoch et al., J. Gen. Viral. (1988) 69:1531-
1574, for a
discussion of the various HSV-1 encoded proteins; U.S. Patent No. 5,171,568
for a
discussion of HSV-1 and HSV-2 gB and gD proteins and the genes encoding
therefor;
Baer et al., Nature (1984) 310:207-211, for the identification of protein
coding sequences
in an EBV genome; and Davison and Scott, Gen. Virol. (1986) 67:1759-1816, for
a
review of VZV.)
[0091] Antigens derived from other viruses will also find use in the
compositions and
methods of the present invention, such as without limitation, proteins from
members of
the families Picomaviridae (e.g., polioviruses, etc.); Caliciviridae;
Togaviridae (e.g.,
rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae;
Bimaviridae;
Rhabodoviridae (e.g., rabies virus, etc.); Filoviridae; Paramyxoviridae (e.g.,
mumps virus,
measles virus, respiratory syneytial virus, etc.); Orthomyxoviridae (e.g.,
influenza virus
types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviridae (e.g., HTLV-
I; HTLV-
II; B1V-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.)), including but not
limited
to antigens from the isolates IRVIEn), HIVsi, HIVLAv, BlVtdu, BIV/vm);
HIV-1; HIV-2; simian immunodeficiency virus (SW) among others. Additionally,
antigens may also be derived from human papillomavirus (HPV) and the tick-
borne
encephalitis viruses. See, e.g. Virology, 3rd Edition (W.K. Joklik ed. 1988);
Fundamental Virology, 2nd Edition (B.N. Fields and D.M. Knipe, eds. 1991), for
a
description of these and other viruses.
[0092] More particularly, the gp120 or gp140 envelope proteins from any of the
above
HIV isolates, including members of the various genetic subtypes of HIV, are
known and
reported (see, e.g., Myers et al., Los Alamos Database, Los Alamos National
Laboratory,
Los Alamos, New Mexico (1992); Myers et al., Human Retroviruses and Aids,
1990, Los
Alamos, New Mexico: Los Alamos National Laboratory; and Modrow et al., J.
Viral.
(1987) 61:570-578, for a comparison of the envelope sequences of a variety of
HIV
isolates) and antigens derived from any of these isolates will find use in the
present
methods. Furthermore, the invention is equally applicable to other immunogenic
proteins
derived from any of the various HIV isolates, including any of the various
envelope
proteins such as gp160 and gp41, gag antigens such as p24gag and p55gag, as
well as
proteins derived from the poi and tat regions.
22
CA 02765058 2012-01-13
[0093] Influenza virus is another example of a virus for which the present
invention will
be particularly useful. Specifically, the envelope glycoproteins HA and NA of
influenza
A are of particular interest for generating an immune response. Numerous HA
subtypes
of influenza A have been identified (Kawaoka et al., Virology (1990) 179:759-
767;
Webster et al., "Antigenic variation among type A influenza viruses," p. 127-
168. In: P.
Palese and D.W. Kingsbury (ed.), Genetics of influenza viruses. Springer-
Verlag, New
York). Thus, proteins derived from any of these isolates can also be used in
the
compositions and methods described herein.
[0094] The compositions and methods described herein will also find use with
numerous
bacterial antigens, such as those derived from organisms that cause diphtheria
(further
discussed above), cholera, anthrax, tuberculosis, tetanus (further discussed
above),
pertussis, meningitis, and other pathogenic states, including, without
limitation,
Bordetella pertussis, Neisseria meningitides (A, B, d, Y), Neisseria
gonorrhoeae,
Helicobacter pylori, and Haemophilus influenza. Hemophilus influenza type B
(H16),
Helicobacter pylori, and combinations thereof. Examples of antigens from
Neisseria
meningitides B are disclosed in the following co-owned patent applications:
PCT/US99/09346; PCT IB98/01665; and PCT 1B99/00103. Examples of parasitic
antigens include those derived from organisms causing malaria and Lyme
disease.
[0095] Further antigens include antigens directed to plague, Rocky Mountain
spotted
fever, smallpox, typhoid, typhus, feline leukemia virus, and yellow fever.
[0096] Additional antigens for use with the invention, which are not
necessarily exclusive
of those listed elsewhere in this application, include the following: (a) a
protein antigen
from N. meningitidis serogroup B, such as those in Refs. 1 to 7 below; (b) an
outer-
membrane vesicle (OMV) preparation from N. meningitidis serogroup B, such as
those
disclosed in Refs. 8, 9, 10, 11, etc. below; (c) a saccharide antigen from N.
meningitidis
serogroup A, C, W135 and/or Y, such as the oligosaccharide disclosed in Ref.
12 below
from serogroup C (see also Ref. 13); (d) a saccharide antigen from
Streptococcus
pneumoniae [e.g. Refs. 14, 15, 16]. (e) an antigen from N. gonorrhoeae [e.g.,
Refs. 1, 2,
3]; (e) an antigen from Chlamydia pneumoniae [e.g., Refs. 17, 18, 19, 20, 21,
22, 23]; (f)
an antigen from Chlamydia trachomatis [e.g. Ref. 24]; (g) an antigen from
hepatitis A
virus, such as inactivated virus [e.g., Refs. 25, 26]; (h) an antigen from
hepatitis B virus,
such as the surface and/or core antigens [e.g., Refs. 26, 27]; (i) an antigen
from hepatitis
23
CA 02765058 2012-01-13
C virus [e.g. Ref. 28]; (j) an antigen from Bordetella pertussis, such as
pertussis holotoxin
(PT) and filamentous haemaglutinin (FHA) from B. pertussis, optionally also in
combination with pertactin and/or agglutinogens 2 and 3 [e.g., Refs. 29 & 30];
(k) a
diphtheria antigen, such as diphtheria toxoid [e.g., chapter 3 of Ref. 31]
e.g. the CRM197
mutant [e.g., Ref. 32] (further discussed above); (1) a tetanus antigen, such
as a tetanus
toxoid [e.g., chapter 4 of Ref. 31] (further discussed above); (m) a protein
antigen from
Helicobacter pylori such as CagA [e.g. Ref. 33], VacA [e.g. Ref. 33], NAP
[e.g. Ref. 34],
HopX [e.g. Ref. 35], HopY [e.g. Ref. 35] and/or urease; (n) a saccharide
antigen from
Haemophilus influenzae B [e.g. Ref. 13]; (o) an antigen from Porphyramonas
gingivalis
[e.g. Ref. 36]; (p) polio antigen(s) [e.g. Refs. 37, 38] such as PPV or OPV;
(q) rabies
antigen(s) [e.g. Ref. 39] such as lyophilized inactivated virus [e.g. Ref. 40,
Rabavertim);
(r) measles, mumps and/or rubella antigens [e.g., chapters 9, 10 and 11 of
Ref. 31]; (s)
influenza antigen(s) [e.g. chapter 19 of Ref. 31], such as the haemagglutinin
and/or
'
neuraminidase surface proteins; (t) an antigen from Moraxella catarrhalis
[e.g., time 41];
(u) an antigen from Streptococcus agalactiae (Group B streptococcus) [e.g.
Refs. 42,43];
(v) an antigen from Streptococcus pyogenes (Group A streptococcus) [e.g. Refs.
43,44,
45]; (w) an antigen from Staphylococcus aureus [e.g. Ref. 46]; and (x)
compositions
comprising one or more of these antigens. Where a saccharide or carbohydrate
antigen is
used, it is preferably conjugated to a carrier protein in order to enhance
immunogenicity
[e.g. Refs. 47 to 56]. Preferred carrier proteins are bacterial toxins or
toxoids, such as
diphtheria or tetanus toxoids. The CRM197 diphtheria toxoid is particularly
preferred.
Other suitable carrier proteins include N. meningitidis outer membrane protein
[e.g. Ref.
57], synthetic peptides [e.g. Refs. 58, 59], heat shock proteins [e.g. Ref.
60], pertussis
proteins [e.g. Refs. 61, 62], protein D from H. Influenzae [e.g. Ref. 63],
toxin A or B
from C. difficile [e.g. Ref. 64], etc. Where a mixture comprises capsular
saccharides from
both serogroups A and C, 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).
Saccharides from
different serogroups of N. meningitidis may be conjugated to the same or
different carrier
proteins. Any suitable conjugation reaction can be used, with any suitable
linker where
necessary. Toxic protein antigens may be detoxified where necessary (e.g.
detoxification
of pertussis toxin by chemical and/or means [Ref. 30]. See: International
patent
application 99/24578 [Ref. 1]; International patent application W099/36544
[Ref. 2];
24
CA 02765058 2012-01-13
International patent application W099/57280 [Ref. 3]; International patent
application
W000/22430 [Ref. 4]; Tettelin et al., (2000) Science 287:1809-1815 [Ref. 5];
International patent application W096/29412 [Ref. 6]; Pizza el al. (2000)
Science
287:1816-1820 [Ref. 7]; International patent application PCT/IB01/00166 [Ref.
8]; Bjune
et al. (1991) Lancet 338(8775):1093-1096 [Ref. 9]; Fulcasawa et al. (1990)
Vaccine
17:2951-2958 [Ref. 10]; Rosenqvist et at. (1998) Dev. Biol. Stand 92:323-333
[Ref. 11];
Costantino et at. (1992) Vaccine 10:691-698 [Ref. 12]; Costantino et al.
(1999) Vaccine
17:1251-1263 [Ref. 13]; Watson (2000) Padiatr Infect Dis J19:331-332 [Ref.
14]; Rubin
(2000) Pediatr Clin North Am 47:269-285, v [Ref. 15]; Jedrzejas (2001)
Microbiol Mol
Biol Rev 65:187-207 [Ref. 16]; International patent application filed on 3rd
July 2001
claiming priority from GB-0016363.4 [Ref. 17]; Kalman et at. ( 1999) Nature
Genetics 21
:385-389 [Ref. 18]; Read et al. (2000) Nucleic Acids Res 28:1397-406 [Ref.
19]; Shirai et
al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527 [Ref. 20]; International
patent
application W099/27105 [Ref. 21]; International patent application W000/27994
[Ref.
22]; International patent application W000/37494 [Ref. 23]; International
patent
application W099/28475 [Ref. 24]; Bell (2000) Pediatr Infect Dis J19:1187-1188
[Ref.
25]; Iwarson (1995) APMIS 103:321-326 [Ref. 26]; Gerlich et al. (1990) Vaccine
8
Suppl:S63-68 & 79-80 [Ref. 27]; Hsu etal. (1999) Clin Liver Dis 3:901-915
[Ref. 28];
Gustafsson et al. (1996) N. EngL J. Med. 334:349-355 [Ref. 29]; Rappuoli et
al. (1991)
TIBTECH 9:232-238 [Ref. 30]; Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-
7216-
1946-0 [Ref. 31]; Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-
70 [Ref.
32]; International patent application W093/18150 [Ref. 33]; International
patent
application W099/53310 [Ref. 34]; International patent application W098/04702
[Ref.
35]; Ross et al. (2001) Vaccine 19:4135-4142 [Ref. 36]; Sutter et at. (2000)
Pediatr Clin
North Am 47:287-308 [Ref. 37]; Zimmerman & Spann (1999) Am Fam Physician
59:113-
118, 125-126 [Ref. 38]; Dreesen (1997) Vaccine 15 Suppl:S2-6 [Ref. 39]; MPIWR
Morb
Mortal Wkly Rep 1998 Jan 16;47(1):12, 19 [Ref. 40]; McMichael (2000) Vaccine
19
Suppl 1:S101-107 [Ref. 41]; Schuchat (1999) Lancet 353(9146):51-6 [Ref. 42];
GB
patent applications 0026333.5, 0028727.6 & 0105640.7 [Ref. 43]; Dale (1999)
Infect Dis
Clin North Am 13:227-43, viii [Ref. 44]; Ferretti etal. (2001) PNAS USA
98:4658-4663
[Ref. 45]; Kuroda et at. (2001) Lancet 357(9264):1225-1240; see also pages
1218-1219
[Ref. 46]; Ramsay et al. (2001) Lancet 357(9251):195-196 [Ref. 47]; Lindberg
(1999)
CA 02765058 2012-01-13
Vaccine 17 Suppl 2:S28-36 [Ref. 48]; Buttery & Moxon (2000) J R Coll
Physicians
London 34:163-168 [Ref. 49]; Ahmad & Chapnick (1999) Infect Dis Clin North Am
13:113-133, vii [Ref. 50]; Goldblatt(1998)J. Med. Microbial. 47:563-567 [Ref.
51];
European patent 0 477 508 [Ref. 52]; US Patent No. 5,306,492 [Ref. 53];
International
patent application W098/42721 [Ref. 54]; Conjugate Vaccines (eds. Cruse et
al.) ISBN
3805549326, particularly vol. 10:48-114 [Ref. 55]; Hermanson (1996)
Bioconjugate
Techniques ISBN: 0123423368 & 012342335X [Ref. 56]; European patent
application
0372501 [Ref. 57]; European patent application 0378881 [Ref. 58]; European
patent
application 0427347 [Ref. 59]; International patent application W093/17712
[Ref. 60];
International patent application W098/58668 [Ref. 61]; European patent
application
0471177 [Ref. 62]; International patent application W000/56360 [Ref. 63];
international
patent application W000/61761 [Ref. 64].
3. Supplemental Components
[0097] The immunogenic compositions of the present invention optionally
include a
variety of supplemental components. Such supplemental components include: (a)
pharmaceuticals such as antibiotics and antiviral agents, nonsteroidal
antiinflammatory
drugs, analgesics, vasodilators, cardiovascular drugs, psychotropics,
neuroleptics,
antidepressants, antiparkinson drugs, beta blockers, calcium channel blockers,
bradykinin
inhibitors, ACE-inhibitors, vasodilators, prolactin inhibitors, steroids,
hormone
antagonists, antihistamines, serotonin antagonists, heparin, chemotherapeutic
agents,
antineoplastics and growth factors, including but not limited to PDGF, EGF,
KGF, IGF-1
and IGF-2, FGF, (b) hormones including peptide hormones such as insulin,
proinsulin,
growth hormone, GHRH, LHRH, EGF, somatostatin, SNX-111, BNP, insulinotropin,
ANP, FSH, LH, PSH and hCG, gonadal steroid hormones (androgens, estrogens and
progesterone), thyroid-stimulating hormone, inhibin, cholecYstokinin, ACTH,
CRF,
dynorphins, endorphins, endothelin, fibronectin fragments, galanin, gastrin,
insulinotropin, glucagon, GTP-binding protein fragments, guanylin, the
leukokinins,
magainin, mastoparans, dermaseptin, systemin, neuromedins, neurotensin,
pancreastatin,
pancreatic polypeptide, substance P, secretin, thymosin, and the like, (c)
enzymes, (d)
transcription or translation mediators, (e) intermediates in metabolic
pathways, (f)
immunomodulators, such as any of the various cytokines including interleukin-
1,
26
CA 02765058 2012-01-13
interleulcin-2, interleulcin-3, interleuldn-4, and gamma-interferon, and (g)
supplementary
immunological adjuvants such as those described below.
[0098] Such supplemental components can be, for example, adsorbed on the
surface of
the microparticles, entrapped within the microparticles, dissolved or
dispersed in solution
while unbound to the microparticles, adsorbed to or entrapped within another
group of
microparticles, and so forth.
[0099] Supplementary immunological adjuvants may be used to enhance the
effectiveness of the immunogenic compositions. For example, such immunological
adjuvants may be administered concurrently with the immunogenic compositions
of the
present invention, e.g., in the same composition or in separate compositions.
Alternatively, such adjuvants may be administered prior or subsequent to the
immunogenic compositions of the present invention.
[01001 Supplementary immunological adjuvants include, but are not limited to:
(1)
aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,
aluminum
sulfate, etc., although it is noted that aluminum salts are associated with
local reactions as
discussed above and are therefore less preferred in some embodiments of the
invention;
(2) other oil-in water emulsion formulations (with or without other specific
immunostimulating agents such as mummy! peptides (see below) or bacterial cell
wall
components), such as for example (a) MF59 (International Publication No.
W090/14837;
Chapter 10 in Vaccine design: the subunit an adjuvant approach, Eds. Powell &
Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5%
Span
= 85 (optionally containing various amounts of MTP-PE (see below), although
not
required) formulated into submicron particles using a microfluidizer such as
Model 110Y*
microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane,
0.4%
*=
Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either
rnicrofluidized into a submicron emulsion or vortexed to generate a larger
particle size
emulsion, and (c) Ribi,7 adjuvant system (RAS), (Ribi Immunochem, Hamilton,
MT)
containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall
components
from the group consisting of monophosphorylipid A (MPL), trehalose climycolate
(TDM),
and cell wall skeleton (CWS), preferably MPL +. CWS (DetoxJ) (for a further
discussion
of suitable. submicron oil-in-water emulsions for use herein, see commonly
owned, patent
application no. 09/015,736, filed on January 29, 1998); (3) saponin adjuvants,
such as
*Trade mark
27
=
CA 02765058 2012-01-13
Quil A, or QS21 (e.g., StimulonJ (Cambridge Bioscience, Worcester, MA)) may be
used
or particles generated therefrom such as ISCOMs (immunostimulating complexes),
which
ICOMS may be devoid of additional detergent e.g., W000/07621; (4) Complete
Freunds
Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as
interleukins (e.g. IL-1, IL-2, IL-4, IL-5,11-6, IL-7, IL-12 (W099/44636),
etc.),
interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-
CSF),
tumor necrosis factor (TNF), etc.; (6) phospholipid adjuvants, including
lipopolysaccharide and liposaccharide phosphate adjuvants, for example,
monophosphoryl lipid A (MPL), 3-0-deacyIated MPL (3dMPL) e.g. GB-2220221, EP-A-
0689454, optionally in the substantial absence of alum when used with
pneumococcal
saccharides e.g. W000/56358; as well as aminoalkyl glucosamine phosphate
compounds
such as those described in U.S. Patent No. 6,355,257; (7) combinations of
3dMPL with,
for example, QS21 and/or oil-in-water emulsions, e.g., EP-A-0835318, EP-A-
0735898,
EP-A-0761231; (8) oligonucleotides comprising CpG motifs (Roman et al.., Nat.
Med.,
1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et
al., J.
Immunol. 1988, 160, 870-876; Chu et Exp. Med, 1997,
186, 1623-1631; Lipford et
al., Eur. J. Immunol. 1997,27, 2340-2344; Moldoveanu etal., Vaccine, 1988, 16,
1216-
1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA,
1996, 93,
2879-2883: Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery etal., J.
Immunol.,
1996, 156,4570-4575; Halpern et al., Cell. Immunol., 1996, 167, 72-78;
Yamamoto et al.,
Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. lmmunol, 1996,
157,2116-2122;
Messina etal., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol.,
1996, 157,
4918-4925; Yi et at., ./: Immunol., 1996, 157, 5394-5402; Yi at al., J.
Immunol., 1998,
160, 4755-4761; and Yi et al., J. Immunol., 1998, 160, 5898-5906;
International patent
applications W096/02555, W098/16247, W098/18810, W098/40100, W098/55495,
W098/37919 and W098/52581) i.e. containing at least one CG dinuckotide (a
cytosine
nucleotide followed by a guanosine nucleotide), with 5 methylcytosine
optionally being
used in place of cytosine; (9) a polyoxyethylene ether or a polyoxyethylene
ester e.g.
W099/52549; (10) a polyoxyethylene sorbitan ester surfactant in combination
with an
octoxynol (W001/21207) or a polyoxyethylene alkyl ether or ester surfactant in
combination with at least one additional non-ionic surfactant such as an
octoxynol
(W001/21152); (11) a saponin and an immunostimulatory oligonucleotide (e.g., a
CpG
28
CA 02765058 2012-01-13
oligonucleotide) (W000/62800); (12) an itnmunostimulant and a particle of
metal salt
e.g. W000/23105; (13) a saponin and an oil-in-water emulsion e.g. W099/11241;
(14) a
saponin (e.g. QS21) + 3dMPL + 1L-12 (optionally + a sterol) e.g. W098/57659;
(15)
detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera
toxin (CT), a
pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-
K63 (where
lysine is substituted for the wild-type amino acid at position 63) LT-R72
(where arginine
is substituted for the wild-type amino acid at position 72), CT-S109 (where
serine is
substituted for the wild-type amino acid at position 109), and PT-K9/G129
(where lysine
is substituted for the wild-type amino acid at position 9 and glycine
substituted at position
129) (see, e.g., International Publication Nos. W093/13202 and W092/19265);
(16)
aminoalkyl glucosaminide 4-phosphates (AGP's), see, e.g., Johnson, D.A. et
al.; Bioorg.
Med. Chem. Lett., 1999 Aug 2; 9(15):2273-8, (17) imidazoquinolines such as
imiquimod
(R-837) and resiquimod (R-848), see, e.g., Vasilakos, J.P. et al.; Cell.
Immunol. 2000
Aug 25; 204(1):64-74, (18) lipopolysaccharide mimetics (including
monophosphoryl
lipid A mimetics), such as non-saccharide phospholipids (e.g., simplified
lipid A analogs
lacking a disaccharide) described in Hawkins, L.D. et al; J. Pharmacol. Exp.
Then, 2002
Feb.; 300(2):655-61 and U.S. Patent No. 6,290,973; (19) adjuvants comprising
natural or
synthetic double-stranded RNA ("dsRNA"), which is generally made up of
intermittent
riboguanylic acid-ribocytidylic acid ([rG-rC]) and riboadenylic acid-
polriboufidylic acid
arA-r15]) base pairs; for further information see, e.g., commonly owned
PCT/US02/30423; and (20) other substances that act as immunostimulating agents
to
enhance the effectiveness of the composition.
[0101] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-
threonyl-
D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),
N-
acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(11-21-dipalmitoyl-sn-
glycero-3-
huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
[0102] For additional examples of adjuvants, see Vaccine Design, The Subunit
and the
Adjuvant Approach, Powell, M.F. and Newman, M.J, eds., Plenum Press, 1995).
=
29
CA 02765058 2012-01-13
4. Formulation and Administration
[0103] The compositions of the present invention will generally include one or
more
pharmaceutically acceptable excipients. For example, vehicles such as water,
saline,
glycerol, polyethylene glycol, hyaluronic acid, ethanol, etc. may be used.
Other
excipients, such as wetting or emulsifying agents, biological buffering
substances, and the
like, may be present. A biological buffer can be virtually any solution which
is
pharmacologically acceptable and which provides the formulation with the
desired pH,
i.e., a pH in the physiological range. Examples include saline, various
buffers including
phosphate buffers, citrate buffers, borate buffers, succinate buffers, and
histidine buffers,
as well as saline buffer combinations, including phosphate buffered saline,
Tris buffered
saline, Hank's buffered saline, and the like.
[01041 Depending on the final dosage form, other excipients known in the art
can also be
introduced, including binders, disintegrants, fillers (diluents), lubricants,
glidants (flow
enhancers), compression aids, colors, sweeteners, preservatives,
suspensing/dispersing
agents, film formers/coatings, flavors and printing inks.
[01051 Once formulated, the compositions of the invention can be administered
parenterally, e.g., by injection (which may be needleless). The compositions
can be
injected subcutaneously, intraperitoneally, intravenously, intraarterially,
intradennally, or
intramuscularly, for example. Other modes of administration include nasal,
mucosal,
intraocular, rectal, vaginal, oral and pulmonary administration,
suppositories, and
transdermal or transcutaneous applications.
[01061 In some embodiments, the compositions of the present invention can be
used for
site-specific targeted delivery. For example, intravenous administration of
the
compositions can be used for targeting the lung, liver, spleen, blood
circulation, or bone
marrow.
[01071 Treatment may be conducted according to a single dose schedule or a
multiple
dose schedule. A multiple dose schedule is one in which a primary course of
administration may be given, followed by one or more additional doses given at
subsequent time intervals, chosen to maintain and/or reinforce the therapeutic
response.
The dosage regimen will also be, at least in part, determined by the need of
the subject
and be dependent on the judgment of the practitioner.
[01081 As previously indicated, the present invention relates to immunogenic
CA 02765058 2012-01-13
pharmaceutical compositions, which comprise biodegradable polymer
microparticles
having adsorbed thereto toxoid and/or polysaccharide-containing antigens. As
also
indicated, the compositions are applicable to a wide range of vaccines,
including vaccines
directed to a wide array of pathogens or tumors.
[0109] Therefore, beneficial compositions include those containing the
following
antigens, either separately or in various combinations: tetanus antigen (e.g.,
tetanus toxoid
antigen), diphtheria antigen (e.g., diphtheria toxoid antigen), hepatitis
antigens (including
HAV, HBV, HCV, HDV, HEY and HGV antigens), varicella virus (chickenpox)
antigens, measles antigens, mumps antigens, rubella antigens, influenza
antigens,
meningococcal antigens (including meningitis A, meningitis B, meningitis C,
meningitis
W and meningitis Y antigens), diphtheria antigens, pertussis antigens, tetanus
antigens,
Hib antigens, and pneumococcal antigens.
[0110] Numerous such antigens are presently available, for example: (A)
Recombinant
DNA hepatitis B antigens (HbsAg) are available, which are made by inserting a
portion
of the HBV genome into yeast. (B) varicella zoster virus antigens are
available,
commonly prepared from lyophilized, live, attenuated varicella virus,
designated the Oka
strain. (C) Polio virus antigens are available, corresponding to either
inactivated or live
poliovirus, with inactivated poliovirus (IPV) antigens typically being
preferred. IPV
antigens are typically formalin-inactivated products, which are produced on
cells, e.g.,
Vero cells or human diploid cells, and commonly correspond to three types of
wild
poliovirus. (D) Live measles virus antigens are frequently prepared from
Edmonston B
strains that have been further attenuated from the original strain (e.g.,
Moraten,
Edmonston-Zagreb, Schwarz and Connaught strains). Measles antigens are
commonly
prepared in chick fibroblast cell cultures or in human fibroblasts. (E) Mumps
virus
antigens are typically live, attenuated virus antigens. They are frequently
prepared from
the Jeryl Lynn attenuated virus strain and are frequently grown in chick
embryo cell
culture. (F) Rubella virus antigens are also typically live, attenuated virus
antigens. One
presently known rubella virus antigen corresponds to live attenuated virus
strain RA 27/3,
and is prepared in human diploid cell culture. (G) Influenza antigen is
frequently
prepared from influenza viruses propagated in chicken embryos. The virus is
inactivated,
purified and treated with an organic solvent to remove surface glycoproteins.
The
antigens are commonly selected from two strains of influenza A and one strain
of
31
CA 02765058 2012-01-13
influenza B. The virus strains chosen for inclusion in influenza vaccine are
typically
reviewed annually to ensure that they include antigens that are expected to
provide the
best protection during the following winter. Influenza antigens also include
live
attenuated virus antigens and antigens derived from tissue culture. (H)
Available
meningococcal antigens include purified capsular polysaccharide antigens (Men-
Ps) and
protein-polysaccharide conjugate antigens (Men-conjugate), including antigens
in which
0-acetylated C-polysaccharide is conjugated to the protein CRM197 (Cross
Reacting
Material 197), and antigens in which de-O-acetylated C-polysaccharide is
conjugated to
tetanus toxoids. (I) Available pertussis antigens include both whole cell and
acellular
pertussis antigens. Acellular antigens have been developed to reduce the
frequency and
severity of both local and systemic adverse reactions associated with whole-
cell pertussis
antigens. Acellular pertussis antigens include, for example, pertussis toxoid,
filamentous
hemagglutinin and pertactin. (J) Available Hib antigens include polysaccharide
antigens
and polysaccharide-protein conjugate antigens, which can have the advantage of
producing greater immune responses in infants and young children relative to
purified
polysaccharide antigen. Available Hib conjugate antigens differ in a number of
ways,
including the protein and the polysaccharide size. Examples include HbOC, PRP-
OMP,
PRP-T and PRP-D antigens. (K) Pneumococcal antigens are typically available as
polysaccharide antigens or as conjugate antigens. An available polysaccharide
pneumococcal vaccine, contains capsular polysaccharide antigens from each of
23 types
of pneumococci (i.e., 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,
15B, 17F, 18C,
19A, 19F, 20, 22F, 23F and 33F, Danish nomenclature). An available
pneumococcal
conjugate vaccine (PCV) contains purified polysaccharides of the capsular
antigens of
seven S. pneumoniae serotypes (serotypes 4, 9V, 14, 18C, 19F, 23F and 6B),
individually
conjugated to CRM197.
[0111] Examples of antigen combinations for use in the present invention
include all
possible combinations of the above, for example, all possible combinations of
DT, DPT,
Hib, Hep, PV, Men, Pnu, Var and MMR, some specific examples of which follow:
32
CA 02765058 2012-01-13
DT
DT-Hep
DT-Men
DT-Hep-Men
DPT
DPT-Hib
DPT-Hep
DPT-PV
DPT-Pnu
DPT-Men
DPT-MIAR
DPT-Var
DPT-Hib-Hep
DPT-Hib-PV
DPT-Hib-Pnu
DPT-Hib-Var
DPT-Hib-MMR
DPT-Hep-PV
DPT-Hep-Pnu
DPT-Hep-Var
DPT-Hep-MMR
DPT-PV-Pnu =
DPT-PV-Var
DPT-PV-MIVIR
DPT-Men-Hib
DPT-Men-Hep
DPT-Men-PV
DPT-Men-Pnu
DPT-Men-Var
DPT-Men-MMR
DPT-Var-MMR
DPT-Var-Pnu
DPT-MMR-Pnu
DPT-Hib-Hep-PV
DPT-Hib-Hep-Pnu
DPT-Hib-Hep-MMR
DPT-Hib-Hep-Var
DPT-Hib-PV-Pnu
DPT-Hep-PV-Pnu
DPT-Hib-PV-MMR
DPT-Hib-PV-Var
DPT-Men-Hib-Hep
DPT-Men-Hib-PV
DPT-Men-Hib-Pnu
33
CA 02765058 2012-01-13
DPT-Men-Hib-MMR
DPT-Men-Hib-Var
DPT-Men-Hep-PV
DPT-Men-Hep-Pnu
DPT-Men-Hep-MMR
DPT-Men-Hep-Var
DPT-Men-PV-Pnu
DPT-Men-PV-MMR
DPT-Men-PV-Var
DPT-Hib-Var-Pnu
DPT-Hib-Var-MMR
DPT-Hep-Var-PV
DPT-Hep-Var-Pnu
DPT-Hep-Var-MMR
DPT-Men-Var-Pnu
DPT-Men-Var-MMR
DPT-PV-Var-Pnu
DPT-PV-Var-MMR
DPT-Pnu-Var-MMR
DPT-Hib-Pnu-MMR
DPT-Hep-PV-MMR
DPT-Hep-Pnu-MMR
DPT-Men-Pnu-MMR.
DPT-PV-Pnu-MMR
DPT-Hib-Hep-PV-Pnu
DPT-Hep-PV-Pnu-Men
DPT-Hib-PV-Pnu-Men
DPT-Hib-Hep-Pnu-Men
DPT-Hib-Hep-PV-Men
DPT-Hib-Hep-PV-Pnu-Men
DPT-Hib-Hep-PV-Pnu-Men-Var-MMR
where, DT=diphtheria toxoid antigen and tetanus toxoid antigen; DTP=diphtheria
toxoid
antigen, tetanus toxoid antigen, and pertussis antigen (including whole-cell
and acellular
pertussis antigens, typically acellular); Hib=Haemophilus influenzae type b
antigen
(including polysaccharide and conjugate antigens); Hep=hepatitis antigen
(including
HAY antigen, HBV antigen, HCV antigen, HDV antigen, HEV antigen, HGV antigen,
and combinations thereof, e.g., HAV antigen-HBV antigen); PV= polio antigen
(including inactivated or live antigens, e.g., inactivated antigens from three
polio strains);
Men=meningococcal (Neisseria meningitidis) antigen (including conjugate and
34
CA 02765058 2012-01-13
polysaccharide antigens, typically conjugate antigens, and including antigens
from Men
A, B, C, W, Y and combinations, e.g., Men A,C antigens, Men A,B,C antigens,
Men
A,C,W,Y antigens, and Men A,B,C,W,Y antigens); Pnu=pneumococcal (Streptococcus
pneumoniae) antigen (including conjugate and polysaccharide antigens,
typically
conjugate antigens); Var=Varicella zoster virus antigen (for example, live,
attenuated
varicella virus); MMR=measles, mumps and rubella antigens (for example, live,
attenuated measles, mumps and rubella virus antigens).
[0112] Recommended administration schedules for vaccines containing several of
the
above antigens are available. As a specific example, the U.S. Department of
Health and
Human Services, Centers for Disease Control and Prevention, National
Immunization
Program, has published its recommended childhood and adolescent immunization
schedules. The present schedules for the United States 2003 are illustrated,
in part, in
Fig. 1 and is summarized below:
= [0113] The schedules to follow are based on Recommended Childhood and
Adolescent
Immunization Schedule--United States 2003, from the U.S. Department of Health
and
Human Services, Centers for Disease Control and Prevention, National
Imm1lni7stion
Program.
[0114] Hepatitis B. (A) First Dose. Birth to two months. Children whose
mothers are
hepatitis B surface antigen positive or whose hepatitis B surface antigen
status is
unknown are recommended to get this dose within 12 hours of birth. All infants
are
recommended to receive the first dose of hepatitis B vaccine soon after birth
and before
hospital discharge; the first dose may also be given by age 2 months if the
infant's mother
is hepatitis B surface antigen negative. Only monovalent hepatitis B vaccine
is presently
recommended for the birth dose. Monovalent or combination vaccine containing
Hep B
may be used to complete the series; four doses of vaccine may be administered
if
combination vaccine is used. Infants born to hepatitis B surface antigen
positive mothers
are recommended to receive hepatitis B vaccine and 0.5 milliliters of
hepatitis B immune
globulin (H BIG) within 12 hours of birth at separate sites. Infants born to
mothers whose
hepatitis B surface antigen status is unknown are recommended to receive the
first dose of
the hepatitis B vaccine series within 12 hours of birth. Maternal blood is
recommended to
be drawn at the time of delivery to determine the mother's hepatitis B surface
antigen
status; if the test is positive, the infant is recommended to receive H BIG as
soon as
CA 02765058 2012-01-13
possible (and no later than one week). (B) Second Dose. One to four months,
but at least
4 weeks after the first dose. The second dose is recommended to be given at
least 4
weeks after the first dose. Hib-containing vaccine is not recommended to be
administered
before age 6 weeks. For infants born to hepatitis B surface antigen positive
mothers, the
second dose is recommended at age 1-2 months and the vaccination series is
recommended to be completed (third or fourth dose) at age 6 months. (c) Third
Dose: six
to 18 months, but at least 16 weeks after the first dose and at least 8 weeks
after the
second dose. In general, it is recommended that the last dose in the
vaccination series
(third or fourth dose) is not to be administered before age 6 months. However,
for infants
born to hepatitis B surface antigen positive mothers, the vaccination series
is
recommended to be completed (third or fourth dose) at age 6 months. (D) Catch-
up
schedule for children age 4 months through 6 years. All children and
adolescents who
have not been immunized against hepatitis B are recommended to begin the Hep B
vaccination series during any visit. Providers are recommended to make special
efforts to
immunize children who were born in, or whose parents were born in, areas of
the world
where hepatitis B virus infection is moderately or highly endemic. Minimum
Interval
Between Dose One to Dose Two: 4 weeks. Minimum Interval Between Dose Two to
Dose Three: 8 weeks (and 16 weeks after first dose). (E) Catch-up schedule for
children
age 7 through 18 years. Minimum Interval Between Dose One to Dose Two: 4
weeks.
Minimum Interval Between Dose Two to Dose Three: 8 weeks (and 16 weeks after
first
dose).
[0115] Diphtheria, Tetanus, Pertussis (DTaP). (A) First Dose: Two months. (B)
Second Dose: Four months. (C) Third Dose: Six months. (D) Fourth Dose: 15 to
18
months. The fourth dose of DTaP may be administered as early a's age 12
months,
provided 6 months have elapsed since the third dose, and the child is unlikely
to return at
age 15 to 18 months. (E) Fifth Dose: 4 to 6 years. (F) Catch-up schedule for
children age
4 months through 6 years. Minimum Interval Between Dose One to Dose Two: 4
weeks.
Minimum Interval Between Dose Two to Dose Three: 4 weeks. Minimum Interval
Between Dose Three to Dose Four: 6 months. Minimum Interval Between Dose Four
to
Dose Five: 6 months. The fifth dose is not necessary if the fourth dose was
given after the
4th birthday.
[0116] Tetanus and Diphtheria. (A) Recommended at age 11 to 12 years if at
least 5
36
CA 02765058 2012-01-13
years have elapsed since the last dose of tetanus and diphtheria toxoid-
containing vaccine.
Subsequent routine Td boosters are recommended every 10 years. (B) Catch-up
schedule
for children age 7 through 18 years. Minimum Interval Between Dose One to Dose
Two:
4 weeks. Minimum Interval Between Dose Two to Dose Three: 6 months. Minimum
Interval Between Dose Three to Booster Dose: 6 months, if 1st dose given at
age less
than 12 months and if current age less then 11 years; 5 years, if 1st dose
given at age 12
months or older and if 3rd dose given at less than 7 years of age and current
age 11 or
older; 10 years, if 3rd dose given at age 7 years or older. (Note: for
children age 7 to 10
years, the interval between the third and booster dose is determined by the
age when the
first dose was given. For adolescents age 11 to 18 years, the interval is
determined by the
age when the third dose was given.)
[0117] Haemophilia influenzae Type b. (A) First Dose: Two months. (B) Second
Dose: Four months. (C) Third Dose: Six months. If PRP-OMP is administered at
ages 2
and 4 months, a dose at age 6 months is not required. (D) Fourth Dose: 12 to
15 months.
(E) Catch-up schedule for children age 4 months through 6 years. (Note:
Vaccine is not
generally recommended for children age 5 years or older.) Dose one to Dose two
minimum intervals: 4 weeks, if first dose given at age less than 12 months; 8
weeks (as a
fmal dose), if first dose given at age 12 to 14 months; if first dose given at
age 15 months
or older, then no further doses are recommended. Dose two to Dose three
minimum
intervals: 4 weeks, if current age less than 12 months; 8 weeks (as final
dose), if second
dose given at age less than 15 months and if current age is 12 months or
older; if second
dose given at age 15 months or older, no further doses needed. (If current age
is less than
12 months and the first 2 doses were PRP-OW, the third and final doses are
recommended to be given at age 12 to 15 months and at least 8 weeks after the
second
dose.) Dose three to Dose four minimum intervals: 8 weeks (as final dose).
This dose is
only recommended for children age 12 months to 5 years who received 3 doses
before
age 12 months.
[0118] Inactivated Polio. (A) First Dose: Two months. (B) Second Dose: Four
months.
(C) Third Dose: 6 to 18 months. (B) Fourth Dose: 4 to 6 years. E) Minimum
Interval
Between Dose One to Dose Two: 4 weeks. Minimum Interval Between Dose Two to
Dose Three: 4 weeks. Minimum Interval Between Dose Three to Dose Four: 4
weeks. For
children who received an all IPV or all OPV series, a fourth dose is not
necessary if third
37
CA 02765058 2012-01-13
dose was given at age 4 years or greater. If both OPV and WV were given as
part of a
series, a total of four doses are recommended to be given, regardless of the
child's current
age. (D) Catch-up schedule for children age 7 through 18 years. (Note: this
vaccine is not
generally recommended for persons age 18 years or older.) Minimum Interval
Between
Dose One to Dose Two: 4 weeks. Minimum Interval Between Dose Two to Dose
Three:
4 weeks.
[0119] Measles, Mumps, Rubella. (A) First Dose: 12 to 15 months. (B) Second
Dose:
4 to 6 years. The second dose of MMR is recommended routinely at age 4 to 6
years but
may be administered during any visit, provided at least 4 weeks have elapsed
since the
first dose and that both doses are administered beginning at or after age 12
months.
Those who have not previously received the second dose are recommended to
complete
the schedule by the 11 to 12 year old visit. (C) Catch-up schedule for
children age 4
months through 6 years. Minimum age: 12 months. Minimum Interval Between Dose
One to Dose Two: 4 weeks. The second dose of M1VIR is recommended routinely at
age 4
to 6 years, but may be given earlier if desired. (D) Catch-up schedule for
children age 7
through 18 years. Minimum Interval Between Dose One to Dose Two: 4 weeks.
[0120] Varicella. 12 to 18 months. Varicella vaccine is recommended at any
visit at or
after age 12 months for susceptible children (i.e., those who lack a reliable
history of
chickenpox). Susceptible persons 13 years of age or older are recommended to
receive 2
doses, given at least 4 weeks apart. Catch-up schedule for children age 7
through 18
years. Minimum Interval Between Dose One to Dose Two: 4 weeks.
[0121] Pneumococcal Conjugate Vaccine. The heptavalent pneumococcal conjugate
vaccine (PC'V) is recommended for all children aged 2 to 23 months and for
certain
children aged 24 to 59 months. (A) First Dose: Two months. (B) Second Dose:
Four
months. (C) Third Dose: Six months. (D) Fourth Dose: 12 to 15 months. (E)
Catch-up
schedule for children age 4 months through 6 years. (Note: this vaccine is not
generally
recommended for children age 5 years or older.) Dose one to Dose two minimum
intervals: if first dose given at age less than 12 months and if current age
less than 24
months, then 4 weeks; if first dose given at age 12 months or older or if
current age 24 to
59 months, then 8 weeks (as final dose); if first dose given at age 24 months
or older,
then for healthy children, no further doses needed. Dose two to Dose three
minimum
intervals: if second dose given at age less than 12 months, then 4 weeks; if
second dose
38
CA 02765058 2012-01-13
given at age 12 months or older, then 8 weeks (as final dose); if second dose
given at age
24 months or older, then for healthy children, no further doses needed. Dose
three to
Dose four minimum intervals: 8 weeks (as final dose). This dose only necessary
for
children age 12 months to 5 years who received 3 doses before age 12 months.
[0122] Pneumococcal polysaccharide vaccine (PPV). Recommended in addition to
PCV for certain high-risk groups.
[0123] Hepatitis A. The hepatitis A series may be given to children two years
of age or
older. Hepatitis A vaccine is recommended for use in selected states and
regions, and for
certain high-risk groups.
[0124] Influenza. Influenza vaccine is recommended annually for children 6
months of
age or older with certain risk factors (including but not limited to asthma,
cardiac disease,
sickle cell disease, HIV and diabetes), and can be administered to all others
wishing to
obtain immunity. Children 12 years of age or younger are recommended to
receive
vaccine in a dosage appropriate for their age. Children 8 years of age or
younger who are
receiving influenza vaccine for the first time are recommended to receive two
doses
separated by at least 4 weeks.
[0125] These schedules pertain to children through age 18 years. Any dose not
given at
the recommended age is, in general, recommended to be given at a subsequent
time, when
indicated and feasible. Obviously, additional schedules can be established for
the various
vaccines listed.
[0126] In certain embodiments, the administration times for a vaccine in
accordance with
the present invention will be based upon the recommended schedule illustrated
in Fig. 1
or described above. For example, a time for administration of a vaccine in
accordance
with the present invention can be selected based upon the recommended schedule
for a
vaccine that contains one (or more) antigens found within the vaccine of the
present
invention.
[0127] As a specific example, vaccine compositions in accordance with the
present
invention that contain diphtheria, pertussis and tetanus antigens (and
optionally one or
more additional antigens) can be administered at any of the following times:
two months,
four months, six months, 15 to 18 months and 4 to 6 years, among other times.
As
another specific example, a vaccine containing DTP/HepB/Hib/PV/Pnu (or any
combination of these) can be administered at 2 months, 4 months, 6 months, or
15
39
CA 02765058 2012-01-13
months, among others. As another specific example, a vaccine containing
DTP/HepB/PV
(or any combination of these) can be administered at 4-6 years, among others.
As another
specific example, a vaccine containing DTP/HepB/Hib/PV/Pnu/MMRNar (or any
combination of these) can be administered at 15 months, among others. As
another
specific example, vaccine compositions that contain diphtheria and tetanus
antigens (and
optionally one or more additional antigens) can be administered at 11-18 years
(and up),
among others. As another specific example, a vaccine containing DT/HepB can be
administered at 11-18 years (and up), among others. As yet another specific
example, a
vaccine containing DT/HepB/MMR/Var (or any combination of these) can be
administered at 11-18 years (and up) among others. And so forth.
C. Experimental
[0128] Below are examples of specific embodiments for carrying out the present
invention. The examples are offered for illustrative purposes only, and are
not intended
to limit the scope of the present invention in any way.
[0129] Efforts have been made to ensure accuracy with respect to numbers used
(e.g.,
amounts, temperatures, etc.), but some experimental error and deviation
should, of
course, be allowed for.
Example 1
Efficiency of Adsorption of Tetanus Toxoid
and Diphtheria Toxoid to PLG particles
[0130] Microparticles are prepared using a 6% w/v solution of RG503 polymer (a
PLG
Polymer having a 50:50 lactide/glycolide molar ratio and a molecular weight of
approximately 34 kDaltons, available from Boehringer Ingelheim) in methylene
chloride.
ml of this solution is homogenized with 2.5m1PBS using a 10-mm probe of a
' homogenizer (Ultra-Turrax T25 IKA-Labortechnik, Germany) for three
minutes at 23,000
rpm, thereby forming a water-in-oil emulsion. This emulsion is then added to
50 ml of
distilled water containing 6 g/m1 dioctyl sodium sulfosuccinate (DSS)
(available from
Sigma, USA) and homogenized at very high speed using a homogenizer with a 204=
probe (ES-15 Omni International, GA, USA) for 25 minutes in an ice bath. This
resulted
in a water-in-oil-in-water emulsion, which is stirred at 1000rpm for 12 h at
room
CA 02765058 2012-01-13
temperature, allowing the methylene chloride to evaporate. The resulting
microparticles
contain 0.05% DSS wt/wt. The size distribution of the resulting microparticle
suspension
is determined using a particle size analyzer (Master Sizer, Malvern
Instruments, UK), and
is found to be between 0.8 and 1.2 p.m.
[0131] Tetanus and Diphtheria toxoids, TT and DT, are adsorbed to the 0.05%
DSS PLG
particles in various buffers with different pH values. The adsorption is
carried out by
incubating 100mg of the above microparticle suspension with lmg or 0.5mg of DT
or TT,
respectively, in 10mM of buffer overnight, while rocking at 4 C. The buffers
are as
follows: PBS pH 7, phosphate pH 7, Citrate pH 5, Borate pH9, Succinate pH5.5,
Succinate pH 5, and Histidine pH 5.
[0132] The suspension is centrifuged the next day, and the supernatant
evaluated for
concentration of unbound protein by HPLC (to establish % adsorption efficiency
by
depletion). Protein on particles is evaluated by first washing the particles
with water to
remove the unbound protein, followed by lyophilization. The amount of adsorbed
protein
is determined by hydrolyzing 10mg of lyophilized particles with 2m1 0.2N NaOH
and 5%
SDS for 4 hours followed by a BCA proteins assay (to establish % adsorption
efficiency
on particles). The results are presented in Tables IA and 1B below.
Buffer
Target Adsorption Adsorption
= Load efficiency Tr efficiency TT
=
=
wt/wt by depletion on particles
PBS pH 7 1% 0 0
Phosphate pH 7 1% 10 0
Citrate pH5 1% 93 81
Borate pH 9 1% 0 0
Succinate p115.5 1% 60 68
Succinate p115 1% 89 ND
Succinate pH 5 0.5 88 ND
Histidine pH 5 0.5 ND 60
Table 1A.
41
CA 02765058 2012-01-13
Buffer
Target Adsorption Adsorption
Load efficiency DT efficiency DT
wt/wt by depletion on particles
PBS pH 7 1% 0 0
Phosphate pH 7 1% . 0 0
Citrate pH5 1% 70 68
Borate pH 9 1% ND ND
Succinate pH5.5 1% 17 0
Succinate p115 1% 35 ND
Succinate pH 5 0.5 32 ND
Histidine pH 5 0.5 N1D 52
Table 1B.
Example 2
Adsorption and Release of Tetanus Toxoid
and Diphtheria Toxoid to/from PLG particles
[0133] Microparticles are prepared as described in Example 1. DT and IT
proteins are
adsorbed to microparticles at a target load of 0.25%, 0.5% or 1% by incubating
100mg of
the above microparticle suspension with an appropriate amount of DT or TT in
10mM of
Histidine buffer overnight, while rocking at 4 C. The suspension is
centrifuged the next
day, and the supernatant evaluated for concentration of unbound protein by
ITPLC to
establish % adsorption efficiency by depletion. The results are presented in
column 2 of
Table 2 below and in Fig. 2.
[0134] The amount of protein released from microparticles was determined by
incubating
10mg of lyophilized, unwashed particles with lml water and left rocking at 4 C
for one
hour, centrifuged, and the supernatant analyzed for released protein by HPLC
as above.
The results are presented in column 3 of Tables 2A and 2B below and in Fig. 2.
42
CA 02765058 2012-01-13
Formulation % adsorbed by depletion % released in lhour
0.25% DT 74.1 9.7
0.50% DT 68.5 16.1
1.00% DT 67.6 19.8
Table 2A.
Formulation % adsorbed by depletion % released in lhour
0.25% 'IT >96.7 <1.6
0.50% TI' >97 <1.6
1.00% TT 98.6 1.2
Table 2B.
Example 3
Adsorption and Release of meningococcal protein-polysaccharide conjugates
to/from PLG particles (RG503/0.05%DSS)
[0135] Microparticles containing 0.05% DSS wt/wt are prepared as in Example 1
above.
Protein-polysaccharide conjugate meningococcal antigens in which purified
capsular
polysaccharide antigens from either Men-A, Men-B, Men-C or Men-W are adsorbed
to
the microparticles.
[0136] Meningococcal conjugate antigens are adsorbed to microparticles at a
target load
of 1% by incubating 100mg of the microparticles with lmg of meningococcal
conjugate
antigen in 10mM of Histidine buffer overnight, while rocking at 4 C. The
suspension is
centrifuged the next day, and the supernatant evaluated for concentration of
unbound
conjugate antigen by HPLC to establish % adsorption efficiency by depletion.
The results
are presented in column 2 of Table 3 below.
[0137] Conjugate antigen adsorbed to particles is evaluated by first washing
the particles
with water to remove the unbound conjugate antigen, followed by
lyophilization. The
amount of adsorbed conjugate antigen is determined by hydrolysis of the
lyophilized
particles, followed by a BCA proteins assay as above. The results are
presented in
column 3 of Table 3 and in Fig. 3.
[0138] To determine the amount of protein released from particles, an aliquot
of the
particle-antigen suspension is lyophilized without washing, and 10mg of the
lyophilized
particles are then incubated with lml water and left rocking at 4 C for one
hour. The
results are presented in column 4 of Table 3 below and in Fig 3.
43
CA 02765058 2012-01-13
formulation % adsorbed by % adsorbed by % 1hour release
depletion . hydrolysis
MenA-CRM 38 31 60.2
MenC-CRM 29 25 78.1
MenY-CRM 34 40 53.5
MenW-CRM 25 29 60.2
Table 3.
Example 4
Immunogenicity of Tetanus Toxoid cro
and Diphtheria Toxoid (DT) formulated with PLG particles
Mouse study.
[0139] For the study, a PLG/DSS microparticle suspension is prepared as
described in
Example 1. Tetanus and Diphtheria toxoids, DT and TT, are adsorbed to the
microparticles at a target load of 0.5% or 1% by incubating 100mg of the above
microparticle suspension with an appropriate amount of DT or TI' in 1 OnaM of
Histidine
buffer overnight, while rocking at 4 C. Then, each formulation was tested
singly
(PLG/TT or PLG/DTT) or in combination (PLG/DT+ PLG/TT). The equivalent Alum
formulations (Alum/TT or Alum/DTT, and (Alum /DTT + Alum/TT)) were also
included
in the study for comparison. Mice were injected at the tibialis anterior with
50 per leg
on day 0 and day 14. Sera were collected on day 28 by orbital sinus bleed.
EL1SA assay.
[0140] The presence of IgG antibody was determined for each mouse by testing
eight
serial dilutions of serum starting at 1/50 on plates that were coated with DT-
CRM or Ti'
antigen. A positive control and positive mouse serum reference were tested on
each plate
for a system-suitable control. The presence of serum antibodies was detected
with a
second antibody conjugated to Horseradish Perwddase in combination with a
colorimetric
substrate, which adsorbs at 450nm. The titers were defined as the reciprocal
serum
dilution that gave an optical density of 0.5 ELISA absorbency. Titers were
obtained by
interpolation from a four-parameter curve fit of absorbance versus dilution.
The
geometric mean titers (GMT) were calculated and the mice having a titer? of 50
were
44
CA 02765058 2012-01-13
reported as responders. The results are presented in Tables 4A-C below. As can
be seen
from these results: (1) No difference was found in titers elicited with PLG
microparticle
prepared at 0.5% or 1% target load for both antigens IT and DT. (2) The
combination of
both antigens formulated with PLG or Alum enhanced the responses to TT' by ¨2-
fold. (3)
Overall, based on these results, the responses elicited with PLG/TT and PLG/DT
are
comparable to Alum! fl and Alum/DT.
2wp2 DT antibody Titers
GMT LCL UCL
#Responders
PLG/DT (0.5%) 17,277 11,718 25,473
PLG/DT (1.0%) - 23,938 13,184 43,464
PLG/DT + PLG/TT 17,786 9,389 33,695
Alum/DT 54,166 40,022 73,308
Alum/DT + Alum/1".1 39,635 24,696 63,609
LCL/UCL = 95% Confidence Limits = Mean (t x SEM)
All calculations were performed using log transformed titer values; final
geometric mean titers
and 95% Confidence Limits shown were obtained by taking the antilog.
Table 4A.
2wp2 TT antibody Titers
GMT LCL UCL
#Responders
PLG/TT (0.5%) 32,182 20,408 50,750
PLG/T1' (1.0%) 41,431 34,842 49,265
PLG/DT + PLG/TT 61,992 36,882 104,199
Alum/TT 36,860 26,672 50,939
Alum/DT + Alum/TT 70,825 55,538 90,322
LCL/UCL =95% Confidence Limits = Mean (t x SEM)
All calculations were performed using log transformed titer values; final
geometric mean titers
and 95% Confidence Limits shown were obtained by taking the antilog.
Table 4B.
CA 02765058 2012-01-13
Example 5
Immunogenicitv of meningococcal protein-polysaccharide conjugates
Formulated with PLG particles (RG503/0.05%DSS) and Alum
[0141] Microparticles containing 0.05% DSS wt/wt are prepared as in Example 1
above.
Protein-polysaccharide conjugate meningococcal antigens in which purified
capsular
polysaccharide antigens from Men-C are conjugated to either CRM197 diphtheria
toxoid
or to ADH, obtained from Chiron Vaccines, Siena, Italy, are adsorbed to the
microparticles. Meningococcal conjugate antigens are adsorbed to the
microparticles at a
target load of 1.0% by incubating 100 mg of the microparticles with 1.0 mg of
meningococcal conjugate antigen in PBS at pH 7.0 overnight, while rocking at 4
C. Mice
were injected at the tibialis anterior with 50 1 per leg on day 0 and day 14.
Sera were
collected on day 28 by orbital sinus bleed.
= [0142] The presence of IgG antibody was determined for each mouse by
testing eight
serial dilutions of serum starting at 1/50 on plates that were coated with Men-
C ADH or
Men-C CRM antigen. A positive control and positive mouse serum reference were
tested
on each plate for a system-suitable control. The presence of serum antibodies
was
detected with a second antibody conjugated to Horseradish Peroxidase in
combination
with a colorimetric substrate, which adsorbs at 450nm. The titers were defined
as the
reciprocal serum dilution that gave an optical density of 0.5 ELISA
absorbency. Titers
were obtained by interpolation from a four-parameter curve fit of absorbance
versus
dilution. The geometric mean titers (GMT) were calculated for IgG total and
IgGi (for
Men-C CRM). The results are presented in Table 5 below. As can be seen from
these
results, antigen formulated with PLO compared favorably to antigen formulated
with
alum.
MenC ADH MenC CRM MenC CRM
(cutoff: 0.5) (cutoff: 0.5) (cutoff: 0.5)
IgG total IgG total IgG1
Formulation GMT lower upper GMT lower upper GMT lower upper
MenC 205 176 238 1,553 1,427 1,691
33,664 29,782 38,053
CRM/PLG
MenC 82 59 114 1,381 1,244 1,533 33,017
30,372 35,892
CRM/Alum
Table 5.
46
CA 02765058 2012-01-13
[0143] Although preferred embodiments of the subject invention have been
described in
some detail, it is understood that obvious variations can be made without
departing from
the scope of the invention.
=
47
