U.S. patent application number 14/202620 was filed with the patent office on 2014-07-10 for streptococcus vaccine compositions and methods of using the same.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF MICHIGAN. The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to James R. Baker, JR., Whitney A. Dunlap, Jessica A. Knowlton, Paul E. Makidon, Benjamin Swanson.
Application Number | 20140193461 14/202620 |
Document ID | / |
Family ID | 43085359 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140193461 |
Kind Code |
A1 |
Baker, JR.; James R. ; et
al. |
July 10, 2014 |
STREPTOCOCCUS VACCINE COMPOSITIONS AND METHODS OF USING THE
SAME
Abstract
The present invention provides methods and compositions for the
stimulation of immune responses. In particular, the present
invention provides immunogenic nanoemulsion compositions and
methods of using the same for the induction of immune responses
(e.g., innate and/or adaptive immune responses (e.g., for
generation of host immunity against a bacterial species of the
genus Streptococcus (e.g., Streptococcus pneumoniae))).
Compositions and methods of the present invention find use in,
among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
Inventors: |
Baker, JR.; James R.; (Ann
Arbor, MI) ; Makidon; Paul E.; (Webberville, MI)
; Dunlap; Whitney A.; (Ann Arbor, MI) ; Knowlton;
Jessica A.; (Ypsilanti, MI) ; Swanson; Benjamin;
(Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF MICHIGAN |
Ann Arbor |
MI |
US |
|
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
MICHIGAN
Ann Arbor
MI
|
Family ID: |
43085359 |
Appl. No.: |
14/202620 |
Filed: |
March 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13320205 |
Feb 13, 2012 |
8668911 |
|
|
PCT/US10/34999 |
May 14, 2010 |
|
|
|
14202620 |
|
|
|
|
61178344 |
May 14, 2009 |
|
|
|
Current U.S.
Class: |
424/244.1 |
Current CPC
Class: |
A61K 2039/55566
20130101; A61K 2039/521 20130101; A61K 39/092 20130101; A61P 31/04
20180101 |
Class at
Publication: |
424/244.1 |
International
Class: |
A61K 39/09 20060101
A61K039/09 |
Claims
1. An immunogenic composition comprising a nanoemulsion and an
immunogen, wherein said nanoemulsion comprises a non-ionic
surfactant, ethanol, cetylpyridinium chloride (CPC), oil and water,
and wherein said immunogen comprises killed whole cell
Streptococcus pneumoniae.
2. The immunogenic composition of claim 1, wherein the composition
comprises between 10.sup.6 and 10.sup.8 CFU of killed Streptococcus
pneumonia.
3. The immunogenic composition of claim 1, wherein the composition
comprises 10.sup.6 CFU of killed Streptococcus pneumonia.
4. The immunogenic composition of claim 1, wherein the composition
comprises 10.sup.8 CFU of killed Streptococcus pneumonia.
5. The immunogenic composition of claim 1, wherein the
Streptococcus pneumoniae cells are killed via mixing with said
nanoemulsion.
6. The immunogenic composition of claim 1, wherein the
Streptococcus pneumoniae cells are killed via mixing with
ethanol.
7. An immunogenic composition comprising a nanoemulsion and a
Streptococcus pneumoniae immunogen, wherein said nanoemulsion
comprises a non-ionic surfactant, ethanol, cetylpyridinium chloride
(CPC), oil and water.
8. The immunogenic composition of claim 7, wherein the
Streptococcus pneumoniae immunogen is selected from the group
consisting of Streptococcus pneumoniae polysaccharide,
Streptococcus pneumoniae protein, killed whole cell Streptococcus
pneumonia, and a combination thereof.
Description
[0001] The present application is a divisional of U.S. patent
application Ser. No. 13/320,205, filed on 13 Feb. 2012, which is a
national phase application under 35 U.S.C. .sctn.371 of PCT
International Application No. PCT/US2010/034999, filed on 14 May
2010, which claims priority to U.S. Provisional Patent Application
Ser. No. 61/178,344 filed 14 May 2009, each of which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides methods and compositions for
the stimulation of immune responses. In particular, the present
invention provides immunogenic nanoemulsion compositions and
methods of using the same for the induction of immune responses
(e.g., innate and/or adaptive immune responses (e.g., for
generation of host immunity against a bacterial species of the
genus Streptococcus (e.g., Streptococcus pneumoniae))).
Compositions and methods of the present invention find use in,
among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
BACKGROUND
[0003] Immunization is a principal feature for improving the health
of people. Despite the availability of a variety of successful
vaccines against many common illnesses, infectious diseases remain
a leading cause of health problems and death. Significant problems
inherent in existing vaccines include the need for repeated
immunizations, and the ineffectiveness of the current vaccine
delivery systems for a broad spectrum of diseases.
[0004] In order to develop vaccines against pathogens that have
been recalcitrant to vaccine development, and/or to overcome the
failings of commercially available vaccines due to expense,
complexity, and underutilization, new methods of antigen
presentation must be developed which will allow for fewer
immunizations, more efficient usage, and/or fewer side effects to
the vaccine.
SUMMARY OF THE INVENTION
[0005] The present invention provides methods and compositions for
the stimulation of immune responses. In particular, the present
invention provides immunogenic nanoemulsion compositions and
methods of using the same for the induction of immune responses
(e.g., innate and/or adaptive immune responses (e.g., for
generation of host immunity against a bacterial species of the
genus Streptococcus (e.g., Streptococcus pneumoniae))).
Compositions and methods of the present invention find use in,
among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
[0006] In some embodiments, the present invention provides an
immunogenic composition comprising a nanoemulsion and one or more
Streptococcus antigens (e.g., Streptococcus pneumoniae antigens).
In some embodiments, the nanoemulsion comprises an aqueous phase,
an oil phase, and a solvent. The present invention is not limited
by the type of nanoemulsion composition. Indeed, a variety of
nanoemulsion compositions find use in the present invention
including, but not limited to, those described herein. The present
invention is not limited by the one or more Streptococcus antigens
utilized in the immunogenic compositions and methods of the
invention. Indeed, a variety of Streptococcus antigens may be
utilized including, but not limited to, Streptococcal bacteria
inactivated and/or killed by nanoemulsion (NE), killed and/or
inactivated Streptococcus bacteria (e.g., via mixing with alcohol
(e.g., ethanol)), whole cell lysates of a Streptococcus bacteria,
one or more isolated, purified and/or recombinant Streptococcus
proteins and/or protein fragments, or other type of Streptococcus
antigen described herein. In a preferred embodiment, the
Streptococcus antigen is killed Streptococcus pneumoniae (e.g.,
(See, e.g., Malley et al., (2001) Infect. Immun. 69, 4870-4873;
Malley et al., (2004) Infect. Immun. 72, 4290-4292)). In some
embodiments, a Streptococcus antigen composition comprises one or
more adjuvants (e.g., cholera toxin (CT)). The present invention is
not limited by the strain and/or serotype of Streptococcus
pneumoniae utilized. A number of strains and/or serotypes of
Streptococcus pneumoniae are described herein, each of which finds
use in an immunogenic composition comprising a nanoemulsion and one
or more Streptococcus antigens. In some embodiments, the
immunogenic composition comprises nanoemulsion inactivated bacteria
of the genus Streptococcus (e.g., Streptococcus pneumoniae). In
some embodiments, the nanoemulsion is W.sub.805EC, although the
present invention is not so limited. For example, in some
embodiments, the nanoemulsion is selected from one of the
nanoemulsion formulations described herein. In some embodiments,
the composition comprises between 1-50% nanoemulsion solution,
although greater and lesser amounts also find use in the invention.
For example, in some embodiments, the immunogenic composition
comprises about 1.0%-10%, about 10%-20%, about 20%-30%, about
30%-40%, about 40%-50%, about 50%-60% or more nanoemulsion
solution. In some embodiments, the immunogenic composition
comprises about 10% nanoemulsion solution. In some embodiments, the
immunogenic composition comprises about 15% nanoemulsion solution.
In some embodiments, the immunogenic composition comprises about
20% nanoemulsion solution. In some embodiments, the immunogenic
composition comprises about 12% nanoemulsion solution. In some
embodiments, the immunogenic composition comprises about 8%
nanoemulsion solution. In some embodiments, the immunogenic
composition comprises about 5% nanoemulsion solution. In some
embodiments, the immunogenic composition comprises about 2%
nanoemulsion solution. In some embodiments, the immunogenic
composition comprises about 1% nanoemulsion solution. In some
embodiments, an immunogenic composition (e.g., that is administered
to a subject in order to generate a Streptococcus specific immune
response in the subject) comprises 10.sup.6 colony forming units
(CFU) of killed Streptococcus (e.g., 10.sup.6 CFU of Streptococcus
pneumoniae bacteria prior to killing/inactivation of the bacteria),
although greater (e.g., about 4.times.10.sup.6 CFU,
8.times.10.sup.6 CFU, 1.times.10.sup.7 CFU, 2.times.10.sup.7 CFU,
4.times.10.sup.7 CFU, 8.times.10.sup.7 CFU, 1.times.10.sup.8 CFU,
1.times.10.sup.9 CFU, or more CFU of killed Streptococcus) and
lesser (e.g., about 1.times.10.sup.6 CFU, 5.times.10.sup.5 CFU,
1.times.10.sup.5 CFU, 5.times.10.sup.4 CFU, 1.times.10.sup.4 CFU,
5.times.10.sup.3 CFU, 1.times.10.sup.3 CFU or fewer CFU of killed
Streptococcus) amounts may also be utilized. In some embodiments,
the composition is stable (e.g., at room temperature (e.g., for 12
hours, one day, two days, three days, four days, a week, two weeks,
three weeks, a month, two months, three months, four months, five
months, six months, 9 months, a year or more). In some embodiments,
the immunogenic composition comprises a pharmaceutically acceptable
carrier. The present invention is not limited to any particular
pharmaceutically acceptable carrier. Indeed, any suitable carrier
may be utilized including but not limited to those described
herein. In some embodiments, the immunogenic composition further
comprises an adjuvant. The present invention is not limited to any
particular adjuvant and any one or more adjuvants described herein
find use in a composition of the invention including but not
limited to adjuvants that skew toward a Th1 and/or Th2 type immune
responses described herein. In some embodiments, the immunogen
comprises a Streptococcus product (e.g., including, but not limited
to, a protein, peptide, polypeptide, nucleic acid, polysaccharide,
or a membrane component derived from the Streptococcus). In some
embodiments, the immunogen and the nanoemulsion are combined in a
single vessel.
[0007] In some embodiments, the present invention provides a method
of inducing an immune response to Streptococcus (e.g.,
Streptococcus pneumoniae) in a subject comprising: providing a
subject and an immunogenic composition comprising a nanoemulsion
and an immunogen, wherein the immunogen comprises a Streptococcus
(e.g., Streptococcus pneumoniae) antigen and administering the
composition to the subject under conditions such that the subject
generates a Streptococcus (e.g., Streptococcus pneumoniae) specific
immune response. The present invention is not limited by the route
chosen for administration of a composition of the present
invention. In some preferred embodiments, administering the
immunogenic composition comprises contacting a mucosal surface of
the subject with the composition. In some embodiments, the mucosal
surface comprises nasal mucosa. In some embodiments, inducing an
immune response induces immunity to Streptococcus (e.g.,
Streptococcus pneumoniae) in the subject. In some embodiments, the
immunity comprises systemic immunity. In some embodiments, the
immunity comprises mucosal immunity. In some embodiments, the
immune response comprises altered (e.g., enhanced) cytokine
expression in the subject. In some embodiments, the immune response
comprises an IgG response (e.g., a systemic IgG response) to the
Streptococcus (e.g., Streptococcus pneumoniae) in the subject. In
some embodiments, the Streptococcus (e.g., Streptococcus
pneumoniae) antigenic composition is administered to the subject
under conditions such that between about 10.sup.5 and 10.sup.8
colony forming units (CFU) of Streptococcus (e.g., Streptococcus
pneumoniae) is present in a dose administered to the subject,
although greater (e.g., about 10.sup.9, 10.sup.10, 10.sup.11,
10.sup.12, or more) and lesser (e.g., about 10.sup.4, 10.sup.3,
10.sup.2 or fewer) CFU of Streptococcus (e.g., Streptococcus
pneumoniae) (e.g., killed whole Streptococcus pneumoniae) may also
be utilized. In some embodiments, a nanoemulsion solution is
utilized to inactivate the Streptococcus (e.g., Streptococcus
pneumoniae). In some embodiments, the nanoemulsion comprises
W.sub.805EC. In some embodiments, the immunity protects the subject
from displaying signs or symptoms of disease caused by
Streptococcus (e.g., Streptococcus pneumoniae). In some
embodiments, the immunity protects the subject from challenge with
a subsequent exposure to live Streptococcus (e.g., Streptococcus
pneumoniae). In some embodiments, the composition further comprises
an adjuvant. In some embodiments, the subject is a human. In some
embodiments, inducing an immune response induces immunity to the
Streptococcus (e.g., Streptococcus pneumoniae) in the subject. In
some embodiments, inducing immunity to Streptococcus (e.g.,
Streptococcus pneumoniae) comprises systemic immunity. In some
embodiments, immunity comprises mucosal immunity. In some
embodiments, the immune response comprises altered (e.g.,
increased) cytokine expression in the subject. In some embodiments,
the immune response comprises a systemic IgG response to the
immunogen. In some embodiments, the immune response comprises a
mucosal IgA response to the immunogen. In some embodiments, each
dose comprises an amount of Streptococcus (e.g., Streptococcus
pneumoniae) antigen sufficient to generate an immune response to
the Streptococcus (e.g., Streptococcus pneumoniae). An effective
amount of the Streptococcus (e.g., Streptococcus pneumoniae)
antigen is a dose that need not be quantified, as long as the
amount of Streptococcus (e.g., Streptococcus pneumoniae) antigen
generates an immune response in a subject when administered to the
subject. In some embodiments, when a nanoemulsion of the present
invention is utilized to inactivate Streptococcus (e.g.,
Streptococcus pneumoniae), it is expected that each dose (e.g.,
administered to a subject to induce and immune response)) comprises
between 10 and 10.sup.10 CFU of Streptococcus (e.g., Streptococcus
pneumoniae) per dose; in some embodiments, each dose comprises
between 10.sup.5 and 10.sup.8 CFU of Streptococcus (e.g.,
Streptococcus pneumoniae) per dose; in some embodiments, each dose
comprises between 10.sup.3 and 10.sup.5 CFU of Streptococcus (e.g.,
Streptococcus pneumoniae) per dose; in some embodiments, each dose
comprises between 10.sup.5 and 10.sup.8 CFU of Streptococcus (e.g.,
Streptococcus pneumoniae) per dose; in some embodiments, each dose
comprises 10.sup.5 CFU of Streptococcus (e.g., Streptococcus
pneumoniae) per dose; in some embodiments, each dose comprises
10.sup.6 CFU of Streptococcus (e.g., Streptococcus pneumoniae) per
dose; and in some embodiments, each dose comprises 10.sup.7 CFU of
Streptococcus (e.g., Streptococcus pneumoniae) per dose. In some
embodiments, each dose comprises more than 10.sup.8 CFU of
Streptococcus (e.g., Streptococcus pneumoniae) per dose. In some
preferred embodiments, each dose comprises 10.sup.8 CFU of
Streptococcus (e.g., Streptococcus pneumoniae) per dose.
[0008] The present invention is not limited to any specific
nanoemulsion composition. Indeed, a variety of nanoemulsion
compositions are described herein that find use in the present
invention. Similarly, the present invention is not limited to a
particular oil present in the nanoemulsion. A variety of oils are
contemplated, including, but not limited to, soybean, avocado,
squalene, olive, canola, corn, rapeseed, safflower, sunflower,
fish, flavor, and water insoluble vitamins. The present invention
is also not limited to a particular solvent. A variety of solvents
are contemplated including, but not limited to, an alcohol (e.g.,
including, but not limited to, methanol, ethanol, propanol, and
octanol), glycerol, polyethylene glycol, and an organic phosphate
based solvent. Nanoemulsion components including oils, solvents and
others are described in further detail below.
[0009] In some embodiments, the emulsion further comprises a
surfactant. The present invention is not limited to a particular
surfactant. A variety of surfactants are contemplated including,
but not limited to, nonionic and ionic surfactants (e.g., TRITON
X-100; TWEEN 20; and TYLOXAPOL).
[0010] In certain embodiments, the emulsion further comprises a
cationic halogen containing compound. The present invention is not
limited to a particular cationic halogen containing compound. A
variety of cationic halogen containing compounds are contemplated
including, but not limited to, cetylpyridinium halides,
cetyltrimethylammonium halides, cetyldimethylethylammonium halides,
cetyldimethylbenzylammonium halides, cetyltributylphosphonium
halides, dodecyltrimethylammonium halides, and
tetradecyltrimethylammonium halides. The present invention is also
not limited to a particular halide. A variety of halides are
contemplated including, but not limited to, halide selected from
the group consisting of chloride, fluoride, bromide, and
iodide.
[0011] In still further embodiments, the emulsion further comprises
a quaternary ammonium containing compound. The present invention is
not limited to a particular quaternary ammonium containing
compound. A variety of quaternary ammonium containing compounds are
contemplated including, but not limited to, Alkyl dimethyl benzyl
ammonium chloride, dialkyl dimethyl ammonium chloride, n-Alkyl
dimethyl benzyl ammonium chloride, n-Alkyl dimethyl ethylbenzyl
ammonium chloride, Dialkyl dimethyl ammonium chloride, and n-Alkyl
dimethyl benzyl ammonium chloride.
[0012] In some embodiments, the present invention provides a
vaccine comprising an immunogenic composition comprising
Streptococcus (e.g., Streptococcus pneumoniae) antigen (e.g.,
killed and/or inactivated whole cell Streptococcus (e.g.,
Streptococcus pneumoniae)). In some embodiments, the invention
provides a kit comprising a vaccine, the vaccine comprising a
nanoemulsion and immunogenic composition comprising Streptococcus
(e.g., Streptococcus pneumoniae) antigen, the emulsion comprising
an aqueous phase, an oil phase, and a solvent. In some embodiments,
the kit further comprises instructions for using the kit for
vaccinating a subject against the Streptococcus (e.g.,
Streptococcus pneumoniae).
[0013] In still further embodiments, the present invention provides
a method of inducing immunity to one or more bacteria of the genus
Streptococcus (e.g., Streptococcus pneumoniae) comprising providing
an emulsion comprising an aqueous phase, an oil phase, and a
solvent; and one or more Streptococcus (e.g., Streptococcus
pneumoniae) antigens; combining the emulsion with the one or more
Streptococcus (e.g., Streptococcus pneumoniae) antigens to generate
a vaccine composition; and administering the vaccine composition to
a subject. In some embodiments, administering comprises contacting
the vaccine composition with a mucosal surface of the subject. For
example, in some preferred embodiments, administering comprises
intranasal administration. In some preferred embodiments, the
administering occurs under conditions such that the subject
generates immunity to the one or more bacteria of the genus
Streptococcus (e.g., Streptococcus pneumoniae) (e.g., via
generating humoral immune responses to the one or more
antigens).
[0014] The present invention is not limited by the nature of the
immune response generated (e.g., post administration of an
immunogenic composition. Indeed, a variety of immune responses may
be generated and measured in a subject administered a composition
of the present invention including, but not limited to, activation,
proliferation or differentiation of cells of the immune system
(e.g., B cells, T cells, dendritic cells, antigen presenting cells
(APCs), macrophages, natural killer (NK) cells, etc.); up-regulated
or down-regulated expression of markers and cytokines; stimulation
of IgA, IgM, and/or IgG titers; splenomegaly (e.g., increased
spleen cellularity); hyperplasia, mixed cellular infiltrates in
various organs, and/or other responses (e.g., of cells) of the
immune system that can be assessed with respect to immune
stimulation known in the art. In some embodiments, administering
comprises contacting a mucosal surface of the subject with the
composition. The present invention is not limited by the mucosal
surface contacted. In some preferred embodiments, the mucosal
surface comprises nasal mucosa. In some embodiments, administering
comprises parenteral administration. The present invention is not
limited by the route chosen for administration of a composition of
the present invention. In some embodiments, inducing an immune
response induces immunity to the one or more bacteria of the genus
Streptococcus (e.g., Streptococcus pneumoniae) in the subject. In
some embodiments, the immunity comprises systemic immunity. In some
embodiments, the immunity comprises mucosal immunity. In some
embodiments, the immune response comprises altered (e.g.,
increased) cytokine expression in the subject. In some embodiments,
the immune response comprises a systemic IgG response. In some
embodiments, the immune response comprises a mucosal IgA response.
In some embodiments, the composition comprises a 15% nanoemulsion
solution. However, the present invention is not limited to this
amount (e.g., percentage) of nanoemulsion. For example, in some
embodiments, a composition comprises less than 10% nanoemulsion
(e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%). In some embodiments,
a composition comprises more than 10% nanoemulsion (e.g., 15%, 20%,
25%, 30%, 35%, 40%. 45%, 50%, 60% or more). In some embodiments, a
composition of the present invention comprises any of the
nanoemulsions described herein. In some embodiments, the
nanoemulsion comprises W.sub.205EC. In some preferred embodiments,
the nanoemulsion comprises W.sub.805EC. In some embodiments, the
nanoemulsion is X8P. In some embodiments, immunity protects the
subject from displaying signs or symptoms of disease caused by a
bacteria of the genus Streptococcus (e.g., Streptococcus
pneumoniae). In some embodiments, immunity protects the subject
from challenge with a subsequent exposure to a live bacterium of
the genus Streptococcus (e.g., Streptococcus pneumoniae). In some
embodiments, the composition further comprises an adjuvant. The
present invention is not limited by the type of adjuvant utilized.
In some embodiments, the adjuvant is cholera toxin (CT). In some
embodiments, the adjuvant is monophosphoryl lipid A and/or a CpG
oligonucleotide. A number of other adjuvants that find use in the
present invention are described herein. In some embodiments, the
subject is a human. In some embodiments, the immunity protects the
subject from displaying signs or symptoms of a infection with a
bacteria of the genus Streptococcus (e.g., Streptococcus
pneumoniae). In some embodiments, immunity reduces the risk of
infection, disease, and/or sickness upon one or more exposures to a
bacteria of the genus Streptococcus (e.g., Streptococcus
pneumoniae).
DESCRIPTION OF THE DRAWINGS
[0015] The following figures form part of the present specification
and are included to further demonstrate certain aspects and
embodiments of the present invention. The invention may be better
understood by reference to one or more of these figures in
combination with the description of specific embodiments presented
herein.
[0016] FIG. 1 shows the preparation of various single dose
formulations of vaccines using stock WCPAg.
[0017] FIG. 2 shows the distribution of anti-S. pneumoniae antibody
titers at 8 weeks after two vaccinations.
[0018] FIG. 3 shows the distribution of anti-S. pneumoniae antibody
titers at 10 weeks after three vaccinations of A) 7.5 .mu.l or B)
0.1 .mu.l of S. pneumoniae antigen.
[0019] FIG. 4 shows serum anti-S. pneumoniae IgG antibodies in CD-1
mice measured at 11 weeks after primary immunization presented as
endpoint titters (+/-sd).
[0020] FIG. 5 shows results of a post-mortem nasalpharyngeal lavage
that was performed at 12 weeks (1 week following challenge). The
lavagent was plated on selective media and wildtype 6B S.
pneumoniae were enumerated. WCAg=whole cell antigen. Data is
presented as endpoint titters (+/-sd).
[0021] FIG. 6 shows the S. pneumoniae colony count using plate
culture after incubation with W.sub.805EC.
[0022] FIG. 7 identifies subjects and the various compositions
administered to the subjects for experiments conducted during the
development of embodiments of the invention.
[0023] FIG. 8 shows serum anti-S. pneumoniae IgG antibodies in CD-1
mice measured at 8 weeks after primary immunization. Data presented
as endpoint titters (+/-sd).
[0024] FIG. 9 shows results of a post-mortem nasalpharyngeal lavage
performed at 9 weeks (1 week following challenge. The lavagent was
plated on selective media and wildtype 6B S. pneumoniae were
enumerated. Data presented as endpoint titters (+/-sd).
[0025] FIG. 10 shows western blots of pneumococcal antigens probed
with either serum from mice vaccinated with NE-S. pneumo (left) or
Alum-S. pneumo (right). Lane 1 and 5 represent the molecular weight
ladder. Lane 2 represents S. pneumoniae inactivated with NE. Lane 3
represents ethanol inactivated S. pneumoniae. Lane 4 represents
wildtype 6B antigen.
GENERAL DESCRIPTION OF THE INVENTION
[0026] The present invention provides methods and compositions for
the stimulation of immune responses. In particular, the present
invention provides immunogenic nanoemulsion compositions and
methods of using the same for the induction of immune responses
(e.g., innate and/or adaptive immune responses (e.g., for
generation of host immunity against a bacterial species of the
genus Streptococcus (e.g., Streptococcus pneumoniae))).
Compositions and methods of the present invention find use in,
among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
[0027] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
nanoemulsion (NE) compositions stabilize and/or preserve (e.g., for
antigen presentation) important antigenic epitopes (e.g.,
recognizable by a subject's immune system) of a bacteria of the
genus Streptococcus (e.g., Streptococcus pneumoniae)) (e.g.,
stabilize and/or preserve hydrophobic and/hydrophilic components in
the oil and water interface of the emulsion (e.g., thereby
providing one or more immunogens (e.g., stabilized antigens)
against which a subject can mount an immune response). In other
embodiments, because NE formulations penetrate the mucosa through
pores, they may carry antigens/immunogens to the submucosal
location of dendritic cells (e.g., thereby initiating and/or
stimulating an immune response). Although an understanding of the
mechanism is not necessary to practice the present invention and
the present invention is not limited to any particular mechanism of
action, in some embodiments, combining a NE and Streptococcus
(e.g., Streptococcus pneumoniae)) antigen stabilizes and/or
preserves Streptococcus (e.g., Streptococcus pneumoniae))
immunogens and provides a proper immunogenic material for
generation of an immune response. Dendritic cells avidly
phagocytose nanoemulsion (NE) oil droplets and this provides one
mechanism to internalize immunogens (e.g., antigenic proteins or
peptide fragments thereof of Streptococcus (e.g., Streptococcus
pneumoniae) for antigen presentation. While other vaccines rely on
inflammatory toxins or other immune stimuli for adjuvant activity
(See, e.g., Holmgren and Czerkinsky, Nature Med. 2005, 11; 45-53),
NEs have not been shown to be inflammatory when placed on the skin
or mucous membranes in studies on animals and in humans. Thus,
although an understanding of the mechanism is not necessary to
practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
a composition comprising a NE of the present invention (e.g., a
composition comprising NE and one or more Streptococcus (e.g.,
Streptococcus pneumoniae)) antigens may act as a "physical"
adjuvant (e.g., that transports and/or presents immunogenic
compositions (e.g., peptides and/or antigens of Streptococcus
(e.g., Streptococcus pneumoniae)) to the immune system. In some
embodiments, mucosal administration of a composition of the present
invention generates mucosal (e.g., signs of mucosal immunity (e.g.,
generation of IgA antibody titers)) as well as systemic
immunity.
[0028] Both cellular and humoral immunity play a role in protection
against multiple pathogens and both can be induced with the NE
formulations of the present invention. In some embodiments,
administration (e.g., mucosal administration) of a composition of
the present invention to a subject results in the induction of both
humoral (e.g., development of specific antibodies) and cellular
(e.g., cytotoxic T lymphocyte) immune responses (e.g., against
Streptococcus (e.g., Streptococcus pneumoniae)). In some
embodiments, a composition of the present invention (e.g.,
immunogenic composition comprising NE and Streptococcus (e.g.,
Streptococcus pneumoniae)) antigen is used as a vaccine (e.g., an
RSV vaccine).
DEFINITIONS
[0029] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0030] As used herein, the term "microorganism" refers to any
species or type of microorganism, including but not limited to,
bacteria, viruses, archaea, fungi, protozoans, mycoplasma, prions,
and parasitic organisms. The term microorganism encompasses both
those organisms that are in and of themselves pathogenic to another
organism (e.g., animals, including humans, and plants) and those
organisms that produce agents that are pathogenic to another
organism, while the organism itself is not directly pathogenic or
infective to the other organism.
[0031] As used herein the term "pathogen," and grammatical
equivalents, refers to an organism (e.g., biological agent),
including microorganisms, that causes a disease state (e.g.,
infection, pathologic condition, disease, etc.) in another organism
(e.g., animals and plants) by directly infecting the other
organism, or by producing agents that causes disease in another
organism (e.g., bacteria that produce pathogenic toxins and the
like). "Pathogens" include, but are not limited to, viruses,
bacteria, archaea, fungi, protozoans, mycoplasma, prions, and
parasitic organisms.
[0032] The terms "bacteria" and "bacterium" refer to all
prokaryotic organisms, including those within all of the phyla in
the Kingdom Procaryotae. It is intended that the term encompass all
microorganisms considered to be bacteria including Mycoplasma,
Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of
bacteria are included within this definition including cocci,
bacilli, spirochetes, spheroplasts, protoplasts, etc.
[0033] As used herein, the term Streptococcus refers to a genus of
spherical Gram positive bacteria belonging to the phylum Firmicutes
and the lactic acid bacteria group. In general, Streptococci are
oxidase-negative and catalase-negative, and many are facultative
anaerobes. In general, individual species of Streptococcus are
classified based on their hemolytic properties. Alpha hemolysis is
caused by a reduction of iron in hemoglobin, giving it a greenish
color on blood agar. Beta-only hemolysis is complete rupture of red
blood cells, giving distinct, wide, clear areas around bacterial
colonies on blood agar. Other streptococci are labeled as gamma
hemolytic.
[0034] As used herein, the term "fungi" is used in reference to
eukaryotic organisms such as molds and yeasts, including dimorphic
fungi.
[0035] As used herein the terms "disease" and "pathologic
condition" are used interchangeably, unless indicated otherwise
herein, to describe a deviation from the condition regarded as
normal or average for members of a species or group (e.g., humans),
and which is detrimental to an affected individual under conditions
that are not inimical to the majority of individuals of that
species or group. Such a deviation can manifest as a state, signs,
and/or symptoms (e.g., diarrhea, nausea, fever, pain, blisters,
boils, rash, immune suppression, inflammation, etc.) that are
associated with any impairment of the normal state of a subject or
of any of its organs or tissues that interrupts or modifies the
performance of normal functions. A disease or pathological
condition may be caused by or result from contact with a
microorganism (e.g., a pathogen or other infective agent (e.g., a
virus or bacteria)), may be responsive to environmental factors
(e.g., malnutrition, industrial hazards, and/or climate), may be
responsive to an inherent defect of the organism (e.g., genetic
anomalies) or to combinations of these and other factors.
[0036] The terms "host" or "subject," as used herein, refer to an
individual to be treated by (e.g., administered) the compositions
and methods of the present invention. Subjects include, but are not
limited to, mammals (e.g., murines, simians, equines, bovines,
porcines, canines, felines, and the like), and most preferably
includes humans. In the context of the invention, the term
"subject" generally refers to an individual who will be
administered or who has been administered one or more compositions
of the present invention (e.g., a composition for inducing an
immune response).
[0037] As used herein, the terms "inactivating," "inactivation" and
grammatical equivalents, when used in reference to a microorganism
(e.g., a pathogen (e.g., a bacterium or a virus)), refer to the
killing, elimination, neutralization and/or reducing of the
capacity of the microorganism (e.g., a pathogen (e.g., a bacterium
or a virus)) to infect and/or cause a pathological response and/or
disease in a host. For example, in some embodiments, the present
invention provides a composition comprising nanoemulsion
(NE)-inactivated Staphylococci. Accordingly, as referred to herein,
compositions comprising "NE-inactivated Staphylococci," "NE-killed
Staphylococci," NE-neutralized Staphylococci "or grammatical
equivalents refer to compositions that, when administered to a
subject, are characterized by the absence of, or significantly
reduced presence of, Staphylococci replication (e.g., over a period
of time (e.g., over a period of days, weeks, months, or longer))
within the host.
[0038] As used herein, the term "fusigenic" is intended to refer to
an emulsion that is capable of fusing with the membrane of a
microbial agent (e.g., a bacterium or bacterial spore). Specific
examples of fusigenic emulsions are described herein.
[0039] As used herein, the term "lysogenic" refers to an emulsion
(e.g., a nanoemulsion) that is capable of disrupting the membrane
of a microbial agent (e.g., a virus (e.g., viral envelope) or a
bacterium or bacterial spore). In preferred embodiments of the
present invention, the presence of a lysogenic and a fusigenic
agent in the same composition produces an enhanced inactivating
effect compared to either agent alone. Methods and compositions
(e.g., for inducing an immune response (e.g., used as a vaccine)
using this improved antimicrobial composition are described in
detail herein.
[0040] The term "emulsion," as used herein, includes classic
oil-in-water or water in oil dispersions or droplets, as well as
other lipid structures that can form as a result of hydrophobic
forces that drive apolar residues (e.g., long hydrocarbon chains)
away from water and drive polar head groups toward water, when a
water immiscible oily phase is mixed with an aqueous phase. These
other lipid structures include, but are not limited to,
unilamellar, paucilamellar, and multilamellar lipid vesicles,
micelles, and lamellar phases. Similarly, the term "nanoemulsion,"
as used herein, refers to oil-in-water dispersions comprising small
lipid structures. For example, in some embodiments, the
nanoemulsions comprise an oil phase having droplets with a mean
particle size of approximately 0.1 to 5 microns (e.g., about 150,
200, 250, 300, 350, 400, 450, 500 nm or larger in diameter),
although smaller and larger particle sizes are contemplated. The
terms "emulsion" and "nanoemulsion" are often used herein,
interchangeably, to refer to the nanoemulsions of the present
invention.
[0041] As used herein, the terms "contact," "contacted," "expose,"
and "exposed," when used in reference to a nanoemulsion and a live
microorganism, refer to bringing one or more nanoemulsions into
contact with a microorganism (e.g., a pathogen) such that the
nanoemulsion inactivates the microorganism or pathogenic agent, if
present. The present invention is not limited by the amount or type
of nanoemulsion used for microorganism inactivation. A variety of
nanoemulsion that find use in the present invention are described
herein and elsewhere (e.g., nanoemulsions described in U.S. Pat.
Apps. 20020045667 and 20040043041, and U.S. Pat. Nos. 6,015,832,
6,506,803, 6,635,676, and 6,559,189, each of which is incorporated
herein by reference in its entirety for all purposes). Ratios and
amounts of nanoemulsion (e.g., sufficient for inactivating the
microorganism (e.g., virus inactivation)) and microorganisms (e.g.,
sufficient to provide an antigenic composition (e.g., a composition
capable of inducing an immune response)) are contemplated in the
present invention including, but not limited to, those described
herein.
[0042] The term "surfactant" refers to any molecule having both a
polar head group, which energetically prefers solvation by water,
and a hydrophobic tail that is not well solvated by water. The term
"cationic surfactant" refers to a surfactant with a cationic head
group. The term "anionic surfactant" refers to a surfactant with an
anionic head group.
[0043] The terms "Hydrophile-Lipophile Balance Index Number" and
"HLB Index Number" refer to an index for correlating the chemical
structure of surfactant molecules with their surface activity. The
HLB Index Number may be calculated by a variety of empirical
formulas as described, for example, by Meyers, (See, e.g., Meyers,
Surfactant Science and Technology, VCH Publishers Inc., New York,
pp. 231-245 (1992)), incorporated herein by reference. As used
herein where appropriate, the HLB Index Number of a surfactant is
the HLB Index Number assigned to that surfactant in McCutcheon's
Volume 1: Emulsifiers and Detergents North American Edition, 1996
(incorporated herein by reference). The HLB Index Number ranges
from 0 to about 70 or more for commercial surfactants. Hydrophilic
surfactants with high solubility in water and solubilizing
properties are at the high end of the scale, while surfactants with
low solubility in water that are good solubilizers of water in oils
are at the low end of the scale.
[0044] As used herein the term "interaction enhancers" refers to
compounds that act to enhance the interaction of an emulsion with a
microorganism (e.g., with a cell wall of a bacteria (e.g., a Gram
negative bacteria) or with a viral envelope (e.g., Vaccinia virus
envelope)). Contemplated interaction enhancers include, but are not
limited to, chelating agents (e.g., ethylenediaminetetraacetic acid
(EDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and
the like) and certain biological agents (e.g., bovine serum albumin
(BSA) and the like).
[0045] The terms "buffer" or "buffering agents" refer to materials,
that when added to a solution, cause the solution to resist changes
in pH.
[0046] The terms "reducing agent" and "electron donor" refer to a
material that donates electrons to a second material to reduce the
oxidation state of one or more of the second material's atoms.
[0047] The term "monovalent salt" refers to any salt in which the
metal (e.g., Na, K, or Li) has a net 1+ charge in solution (i.e.,
one more proton than electron).
[0048] The term "divalent salt" refers to any salt in which a metal
(e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.
[0049] The terms "chelator" or "chelating agent" refer to any
materials having more than one atom with a lone pair of electrons
that are available to bond to a metal ion.
[0050] The term "solution" refers to an aqueous or non-aqueous
mixture.
[0051] As used herein, the term "a composition for inducing an
immune response" refers to a composition that, once administered to
a subject (e.g., once, twice, three times or more (e.g., separated
by weeks, months or years)), stimulates, generates and/or elicits
an immune response in the subject (e.g., resulting in total or
partial immunity to a microorganism (e.g., pathogen) capable of
causing disease). In preferred embodiments of the invention, the
composition comprises a nanoemulsion and an immunogen. In further
preferred embodiments, the composition comprising a nanoemulsion
and an immunogen comprises one or more other compounds or agents
including, but not limited to, therapeutic agents, physiologically
tolerable liquids, gels, carriers, diluents, adjuvants, excipients,
salicylates, steroids, immunosuppressants, immunostimulants,
antibodies, cytokines, antibiotics, binders, fillers,
preservatives, stabilizing agents, emulsifiers, and/or buffers. An
immune response may be an innate (e.g., a non-specific) immune
response or a learned (e.g., acquired) immune response (e.g. that
decreases the infectivity, morbidity, or onset of mortality in a
subject (e.g., caused by exposure to a pathogenic microorganism) or
that prevents infectivity, morbidity, or onset of mortality in a
subject (e.g., caused by exposure to a pathogenic microorganism)).
Thus, in some preferred embodiments, a composition comprising a
nanoemulsion and an immunogen is administered to a subject as a
vaccine (e.g., to prevent or attenuate a disease (e.g., by
providing to the subject total or partial immunity against the
disease or the total or partial attenuation (e.g., suppression) of
a sign, symptom or condition of the disease.
[0052] As used herein, the term "adjuvant" refers to any substance
that can stimulate an immune response (e.g., a mucosal immune
response). Some adjuvants can cause activation of a cell of the
immune system (e.g., an adjuvant can cause an immune cell to
produce and secrete a cytokine) Examples of adjuvants that can
cause activation of a cell of the immune system include, but are
not limited to, the nanoemulsion formulations described herein,
saponins purified from the bark of the Q. saponaria tree, such as
QS21 (a glycolipid that elutes in the 21st peak with HPLC
fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.);
poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus
Research Institute, USA); derivatives of lipopolysaccharides such
as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,
Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and
threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine
disaccharide related to lipid A; OM Pharma SA, Meyrin,
Switzerland); cholera toxin (CT), and Leishmania elongation factor
(a purified Leishmania protein; Corixa Corporation, Seattle,
Wash.). Traditional adjuvants are well known in the art and
include, for example, aluminum phosphate or hydroxide salts
("alum"). In some embodiments, compositions of the present
invention (e.g., comprising HIV or an immunogenic epitope thereof
(e.g., gp120)) are administered with one or more adjuvants (e.g.,
to skew the immune response towards a Th1 and/or Th2 type
response).
[0053] As used herein, the term "an amount effective to induce an
immune response" (e.g., of a composition for inducing an immune
response), refers to the dosage level required (e.g., when
administered to a subject) to stimulate, generate and/or elicit an
immune response in the subject. An effective amount can be
administered in one or more administrations (e.g., via the same or
different route), applications or dosages and is not intended to be
limited to a particular formulation or administration route.
[0054] As used herein, the term "under conditions such that said
subject generates an immune response" refers to any qualitative or
quantitative induction, generation, and/or stimulation of an immune
response (e.g., innate or acquired).
[0055] A used herein, the term "immune response" refers to a
response by the immune system of a subject. For example, immune
responses include, but are not limited to, a detectable alteration
(e.g., increase) in Toll-like receptor (TLR) activation, lymphokine
(e.g., cytokine (e.g., Th1 or Th2 type cytokines) or chemokine)
expression and/or secretion, macrophage activation, dendritic cell
activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell
activation, and/or B cell activation (e.g., antibody generation
and/or secretion). Additional examples of immune responses include
binding of an immunogen (e.g., antigen (e.g., immunogenic
polypeptide)) to an MHC molecule and inducing a cytotoxic T
lymphocyte ("CTL") response, inducing a B cell response (e.g.,
antibody production), and/or T-helper lymphocyte response, and/or a
delayed type hypersensitivity (DTH) response against the antigen
from which the immunogenic polypeptide is derived, expansion (e.g.,
growth of a population of cells) of cells of the immune system
(e.g., T cells, B cells (e.g., of any stage of development (e.g.,
plasma cells), and increased processing and presentation of antigen
by antigen presenting cells. An immune response may be to
immunogens that the subject's immune system recognizes as foreign
(e.g., non-self antigens from microorganisms (e.g., pathogens), or
self-antigens recognized as foreign). Thus, it is to be understood
that, as used herein, "immune response" refers to any type of
immune response, including, but not limited to, innate immune
responses (e.g., activation of Toll receptor signaling cascade)
cell-mediated immune responses (e.g., responses mediated by T cells
(e.g., antigen-specific T cells) and non-specific cells of the
immune system) and humoral immune responses (e.g., responses
mediated by B cells (e.g., via generation and secretion of
antibodies into the plasma, lymph, and/or tissue fluids). The term
"immune response" is meant to encompass all aspects of the
capability of a subject's immune system to respond to antigens
and/or immunogens (e.g., both the initial response to an immunogen
(e.g., a pathogen) as well as acquired (e.g., memory) responses
that are a result of an adaptive immune response).
[0056] As used herein, the terms "toll receptors" and "TLRs" refer
to a class of receptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,
TLR7, TLR8, TLR9, TLRT0, TLR 11) that recognize special patterns of
pathogens, termed pathogen-associated molecular patterns (See,
e.g., Janeway and Medzhitov, (2002) Annu Rev. Immunol. 20,
197-216). These receptors are expressed in innate immune cells
(e.g., neutrophils, monocytes, macrophages, dendritic cells) and in
other types of cells such as endothelial cells. Their ligands
include bacterial products such as LPS, peptidoglycans,
lipopeptides, and CpG DNA. TLRs are receptors that bind to
exogenous ligands and mediate innate immune responses leading to
the elimination of invading microbes. The TLR-triggered signaling
pathway leads to activation of transcription factors including
NFkB, which is important for the induced expression of
proinflammatory cytokines and chemokines TLRs also interact with
each other. For example, TLR2 can form functional heterodimers with
TLR1 or TLR6. The TLR2/1 dimer has different ligand binding profile
than the TLR2/6 dimer (Ozinsky et al., 2000). In some embodiments,
a nanoemulsion adjuvant activates cell signaling through a TLR
(e.g., TLR2 and/or TLR4). Thus, methods described herein include a
nanoemulsion adjuvant composition (e.g., composition comprising NE
adjuvant optionally combined with one or more immunogens (e.g.,
Streptococcus (e.g., Streptococcus pneumoniae) antigens) that when
administered to a subject, activates one or more TLRs and
stimulates an immune response (e.g., innate and/or
adaptive/acquired immune response) in a subject. Such an adjuvant
can activate TLRs (e.g., TLR2 and/or TLR4) by, for example,
interacting with TLRs (e.g., NE adjuvant binding to TLRs) or
activating any downstream cellular pathway that occurs upon binding
of a ligand to a TLR. NE adjuvants described herein that activate
TLRs can also enhance the availability or accessibility of any
endogenous or naturally occurring ligand of TLRs. A NE adjuvant
that activates one or more TLRs can alter transcription of genes,
increase translation of mRNA or increase the activity of proteins
that are involved in mediating TLR cellular processes. For example,
NE adjuvants described herein that activate one or more TLRs (e.g.,
TLR2 and/or TLR4) can induce expression of one or more cytokines
(e.g., IL-8, IL-12p40, and/or IL-23)
[0057] As used herein, the term "immunity" refers to protection
from disease (e.g., preventing or attenuating (e.g., suppression)
of a sign, symptom or condition of the disease) upon exposure to a
microorganism (e.g., pathogen) capable of causing the disease.
Immunity can be innate (e.g., non-adaptive (e.g., non-acquired)
immune responses that exist in the absence of a previous exposure
to an antigen) and/or acquired/adaptive (e.g., immune responses
that are mediated by B and T cells following a previous exposure to
antigen (e.g., that exhibit increased specificity and reactivity to
the antigen)).
[0058] As used herein, the terms "immunogen" and "antigen" refer to
an agent (e.g., a microorganism (e.g., bacterium, virus or fungus)
and/or portion or component thereof (e.g., a protein antigen)) that
is capable of eliciting an immune response in a subject. In
preferred embodiments, immunogens elicit immunity against the
immunogen (e.g., microorganism (e.g., pathogen or a pathogen
product)) when administered in combination with a nanoemulsion of
the present invention. As used herein, the term Streptococcus
antigen refers to a component or product of a bacteria of the genus
Streptococcus that elicits an immune response when administered to
a subject.
[0059] As used herein, the term "pathogen product" refers to any
component or product derived from a pathogen including, but not
limited to, polypeptides, peptides, proteins, nucleic acids,
membrane fractions, and polysaccharides.
[0060] As used herein, the term "enhanced immunity" refers to an
increase in the level of adaptive and/or acquired immunity in a
subject to a given immunogen (e.g., microorganism (e.g., pathogen))
following administration of a composition (e.g., composition for
inducing an immune response of the present invention) relative to
the level of adaptive and/or acquired immunity in a subject that
has not been administered the composition (e.g., composition for
inducing an immune response of the present invention).
[0061] As used herein, the terms "purified" or "to purify" refer to
the removal of contaminants or undesired compounds from a sample or
composition. As used herein, the term "substantially purified"
refers to the removal of from about 70 to 90%, up to 100%, of the
contaminants or undesired compounds from a sample or
composition.
[0062] As used herein, the terms "administration" and
"administering" refer to the act of giving a composition of the
present invention (e.g., a composition for inducing an immune
response (e.g., a composition comprising a nanoemulsion and an
immunogen)) to a subject. Exemplary routes of administration to the
human body include, but are not limited to, through the eyes
(ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs
(inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g.,
intravenously, subcutaneously, intraperitoneally, etc.), topically,
and the like.
[0063] As used herein, the terms "co-administration" and
"co-administering" refer to the administration of at least two
agent(s) (e.g., a composition comprising a nanoemulsion and an
immunogen and one or more other agents--e.g., an adjuvant) or
therapies to a subject. In some embodiments, the co-administration
of two or more agents or therapies is concurrent. In other
embodiments, a first agent/therapy is administered prior to a
second agent/therapy. In some embodiments, co-administration can be
via the same or different route of administration. Those of skill
in the art understand that the formulations and/or routes of
administration of the various agents or therapies used may vary.
The appropriate dosage for co-administration can be readily
determined by one skilled in the art. In some embodiments, when
agents or therapies are co-administered, the respective agents or
therapies are administered at lower dosages than appropriate for
their administration alone. Thus, co-administration is especially
desirable in embodiments where the co-administration of the agents
or therapies lowers the requisite dosage of a potentially harmful
(e.g., toxic) agent(s), and/or when co-administration of two or
more agents results in sensitization of a subject to beneficial
effects of one of the agents via co-administration of the other
agent. In other embodiments, co-administration is preferable to
elicit an immune response in a subject to two or more different
immunogens (e.g., microorganisms (e.g., pathogens)) at or near the
same time (e.g., when a subject is unlikely to be available for
subsequent administration of a second, third, or more composition
for inducing an immune response).
[0064] As used herein, the term "topically" refers to application
of a compositions of the present invention (e.g., a composition
comprising a nanoemulsion and an immunogen) to the surface of the
skin and/or mucosal cells and tissues (e.g., alveolar, buccal,
lingual, masticatory, vaginal or nasal mucosa, and other tissues
and cells which line hollow organs or body cavities).
[0065] In some embodiments, the compositions of the present
invention are administered in the form of topical emulsions,
injectable compositions, ingestible solutions, and the like. When
the route is topical, the form may be, for example, a spray (e.g.,
a nasal spray), a cream, or other viscous solution (e.g., a
composition comprising a nanoemulsion and an immunogen in
polyethylene glycol).
[0066] The terms "pharmaceutically acceptable" or
"pharmacologically acceptable," as used herein, refer to
compositions that do not substantially produce adverse reactions
(e.g., toxic, allergic or immunological reactions) when
administered to a subject.
[0067] As used herein, the term "pharmaceutically acceptable
carrier" refers to any of the standard pharmaceutical carriers
including, but not limited to, phosphate buffered saline solution,
water, and various types of wetting agents (e.g., sodium lauryl
sulfate), any and all solvents, dispersion media, coatings, sodium
lauryl sulfate, isotonic and absorption delaying agents,
disintrigrants (e.g., potato starch or sodium starch glycolate),
polyethyl glycol, and the like. The compositions also can include
stabilizers and preservatives. Examples of carriers, stabilizers
and adjuvants have been described and are known in the art (See
e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack
Publ. Co., Easton, Pa. (1975), incorporated herein by
reference).
[0068] As used herein, the term "pharmaceutically acceptable salt"
refers to any salt (e.g., obtained by reaction with an acid or a
base) of a composition of the present invention that is
physiologically tolerated in the target subject. "Salts" of the
compositions of the present invention may be derived from inorganic
or organic acids and bases. Examples of acids include, but are not
limited to, hydrochloric, hydrobromic, sulfuric, nitric,
perchloric, fumaric, maleic, phosphoric, glycolic, lactic,
salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,
methanesulfonic, ethanesulfonic, formic, benzoic, malonic,
sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the
like. Other acids, such as oxalic, while not in themselves
pharmaceutically acceptable, may be employed in the preparation of
salts useful as intermediates in obtaining the compositions of the
invention and their pharmaceutically acceptable acid addition
salts.
[0069] Examples of bases include, but are not limited to, alkali
metal (e.g., sodium) hydroxides, alkaline earth metal (e.g.,
magnesium) hydroxides, ammonia, and compounds of formula
NW.sub.4.sup.+, wherein W is C.sub.1-4 alkyl, and the like.
[0070] Examples of salts include, but are not limited to: acetate,
adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide,
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,
persulfate, phenylpropionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate, undecanoate, and the
like. Other examples of salts include anions of the compounds of
the present invention compounded with a suitable cation such as
Na.sup.+, NH.sub.4.sup.+, and NW.sub.4.sup.+ (wherein W is a
C.sub.1-4 alkyl group), and the like. For therapeutic use, salts of
the compounds of the present invention are contemplated as being
pharmaceutically acceptable. However, salts of acids and bases that
are non-pharmaceutically acceptable may also find use, for example,
in the preparation or purification of a pharmaceutically acceptable
compound.
[0071] For therapeutic use, salts of the compositions of the
present invention are contemplated as being pharmaceutically
acceptable. However, salts of acids and bases that are
non-pharmaceutically acceptable may also find use, for example, in
the preparation or purification of a pharmaceutically acceptable
composition.
[0072] As used herein, the term "at risk for disease" refers to a
subject that is predisposed to experiencing a particular disease.
This predisposition may be genetic (e.g., a particular genetic
tendency to experience the disease, such as heritable disorders),
or due to other factors (e.g., environmental conditions, exposures
to detrimental compounds present in the environment, etc.). Thus,
it is not intended that the present invention be limited to any
particular risk (e.g., a subject may be "at risk for disease"
simply by being exposed to and interacting with other people), nor
is it intended that the present invention be limited to any
particular disease.
[0073] "Nasal application", as used herein, means applied through
the nose into the nasal or sinus passages or both. The application
may, for example, be done by drops, sprays, mists, coatings or
mixtures thereof applied to the nasal and sinus passages.
[0074] As used herein, the term "kit" refers to any delivery system
for delivering materials. In the context of immunogenic agents
(e.g., compositions comprising a nanoemulsion and an immunogen),
such delivery systems include systems that allow for the storage,
transport, or delivery of immunogenic agents and/or supporting
materials (e.g., written instructions for using the materials,
etc.) from one location to another. For example, kits include one
or more enclosures (e.g., boxes) containing the relevant
immunogenic agents (e.g., nanoemulsions) and/or supporting
materials. As used herein, the term "fragmented kit" refers to
delivery systems comprising two or more separate containers that
each contain a subportion of the total kit components. The
containers may be delivered to the intended recipient together or
separately. For example, a first container may contain a
composition comprising a nanoemulsion and an immunogen for a
particular use, while a second container contains a second agent
(e.g., an antibiotic or spray applicator). Indeed, any delivery
system comprising two or more separate containers that each
contains a subportion of the total kit components are included in
the term "fragmented kit." In contrast, a "combined kit" refers to
a delivery system containing all of the components of an
immunogenic agent needed for a particular use in a single container
(e.g., in a single box housing each of the desired components). The
term "kit" includes both fragmented and combined kits.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Streptococcus pneumoniae is a Gram-positive bacterium
responsible for considerable morbidity and mortality (particularly
in the young and aged), causing invasive diseases such as
pneumoniae, bacteraemia and meningitis, and diseases associated
with colonization, such as acute Otitis media. The rate of
pneumococcal pneumoniae in the US for persons over 60 years of age
is estimated to be 3 to 8 per 100,000. In 20% of cases this leads
to bacteraemia, and other manifestations such as meningitis, with a
mortality rate close to 30% even with antibiotic treatment.
[0076] Pneumococcus is encapsulated with a chemically linked
polysaccharide which confers serotype specificity. There are 90
known serotypes of pneumococci, and the capsule is the principle
virulence determinant for pneumococci, as the capsule not only
protects the inner surface of the bacteria from complement, but is
itself poorly immunogenic. Polysaccharides are T-independent
antigens, and have been shown to not be processed or presented on
MHC molecules to interact with T-cells. They can however, stimulate
the immune system through an alternate mechanism which involves
cross-linking of surface receptors on B cells. It has been
documents that protection against invasive pneumococci disease is
correlated most strongly with antibody specific for the capsule,
and the protection is serotype specific.
[0077] Streptococcus pneumoniae is the most common cause of
invasive bacterial disease and Otitis media in infants and young
children. Likewise, the elderly mount poor responses to
pneumococcal vaccines (See, e.g., Roghmann et al., (1987), J.
Gerontol. 42:265-270], hence the increased incidence of bacterial
pneumonia in this population (See, e.g., Verghese and Berk, (1983)
Medicine (Baltimore) 62:271-285).
[0078] Accordingly, the present invention provides methods and
compositions for the stimulation of immune responses. In
particular, the present invention provides immunogenic nanoemulsion
compositions and methods of using the same for the induction of
immune responses (e.g., innate and/or adaptive immune responses
(e.g., for generation of host immunity against a bacterial species
of the genus Streptococcus (e.g., Streptococcus pneumoniae))).
Compositions and methods of the present invention find use in,
among other things, clinical (e.g. therapeutic and preventative
medicine (e.g., vaccination)) and research applications.
[0079] In some embodiments, the present invention provides
nanoemulsion adjuvants and compositions comprising the same (e.g.,
vaccines) for the stimulation of immune responses (e.g., immunity)
against a bacterial species of the genus Streptococcus (e.g.,
Streptococcus pneumoniae). In some embodiments, the present
invention provides nanoemulsion adjuvant compositions that
stimulate and/or elicit immune responses (e.g., innate immune
responses and/or adaptive/acquired immune responses) when
administered to a subject (e.g., a human subject)). In some
embodiments, the present invention provides nanoemulsion adjuvant
compositions comprising one or a plurality of Streptococcus (e.g.,
Streptococcus pneumoniae)) antigens (e.g., Streptococcus components
and/or inactivated Streptococcus). The present invention is not
limited to any particular nanoemulsion or Streptococcus (e.g.,
Streptococcus pneumoniae)) antigen. Exemplary immunogenic
compositions (e.g., vaccine compositions) and methods of
administering the compositions are described in more detail
below.
[0080] In some embodiments, the present invention provides an
immunogenic composition comprising a nanoemulsion and one or more
Streptococcus antigens (e.g., Streptococcus pneumoniae antigens).
In some embodiments, the present invention provides a method of
inducing an immune response to Streptococcus (e.g., Streptococcus
pneumoniae) in a subject comprising: providing a subject and an
immunogenic composition comprising a nanoemulsion and an immunogen,
wherein the immunogen comprises a Streptococcus (e.g.,
Streptococcus pneumoniae) antigen and administering the composition
to the subject under conditions such that the subject generates a
Streptococcus (e.g., Streptococcus pneumoniae) specific immune
response. The present invention is not limited by the route chosen
for administration of a composition of the present invention. In
some preferred embodiments, administering the immunogenic
composition comprises contacting a mucosal surface of the subject
with the composition. In some embodiments, the mucosal surface
comprises nasal mucosa. In some embodiments, inducing an immune
response induces immunity to Streptococcus (e.g., Streptococcus
pneumoniae) in the subject.
[0081] Experiments were conducted during development of embodiments
of the invention to determine if a composition comprising a
nanoemulsion (NE) and Streptococcus pneumoniae antigen could be
utilized to generate an immune response in a subject. Nasal
immunization with a whole cell Streptococcus pneumoniae antigen
(WCPAg) mixed with nanoemulsion was performed and shown to induce
an IgG response in a host subject and the ability to eradicate
upper respiratory colonization of S. pneumoniae.
[0082] In particular, as described in Examples 1-4, experiments
were conducted to determine whether S. pneumoniae mixed with
nanoemulsion could produce an immune response. CD-1 and C57/B6 mice
were immunized with three intranasal doses of WCPAg
(1.times.10.sup.8 CFU or 1.times.10.sup.6 CFU) (See, e.g., Malley
et al., (2001) Infect. Immun. 69, 4870-4873; Malley et al., (2004)
Infect. Immun. 72, 4290-4292)) combined with various concentrations
of nanoemulsion. Serum antibody titers show that mixing of
Staphylococcus antigen (WCPAg/W.sub.805EC) with nanoemulsion
resulted in 10- to 20-fold increase in immune response over the
levels obtained without nanoemulsion, and was comparable with the
standard intramuscular immunization with alum adjuvant (See FIGS.
3A and 3B).
[0083] To test the protective effect of immunization mice were
intranasally infected with S. pneumoniae. Analysis of bacteria
recovered from the nasal washes 7 days post-colonization indicated
protection against pneumococcal colonization and decrease in
colonization of the upper respiratory tract in the mice immunized
with 10.sup.8 CFU WCPAg plus nanoemulsion (See FIG. 4).
[0084] The efficacy of intranasal WCPAg/W.sub.805EC vaccine was
evaluated at antigen doses of 7.5 .mu.L and 0.14 in 1, 5, 10 and
20% nanoemulsion (NE). The results indicated that the doses of 7.5
.mu.L WCPAg in 1, 5, 10, and 20% NE elicited an immune response
similar to that of the 7.5 .mu.L WCPAg+Alum group. Mucosal
vaccination with 7.5 .mu.L WCPAg in 1, 5, 10, and 20% NE produced
an immune response greater than 1.times.10.sup.5 mean IgG titers
after three vaccinations. (See, e.g., Examples 2-4). Additionally,
after challenging the mice with 1.times.10.sup.5 colony forming
units (CFU) S. pneumoniae, there was a marked reduction in carriage
in the 7.5 .mu.L WCPAg/NE and 7.5 .mu.L+Alum groups versus control
groups (See FIG. 4). Complete blood counts (white blood cells,
neutrophils and monocytes) showed no abnormalities within any of
the groups.
[0085] Accordingly, in some embodiments, the present invention
provides that administration (e.g., nasal administration) of a
composition comprising nanoemulsion and S. pneumoniae antigen
(e.g., whole cell S. pneumoniae) to a subject produces immunity
toward S. pneumoniae in the subject thereby protecting the subject
against pneumococcal infection. In some embodiments, compositions
and method of the present invention provide Streptococci (e.g., S.
pneumoniae) specific protective immune responses in a subject
(e.g., similar to and/or greater than conventional Streptococci
(e.g., S. pneumoniae) vaccines (e.g., alum-adjuvanted
vaccines))).
[0086] The present invention is not limited by the type of bacteria
of the genus Streptococci utilized in the immunogenic compositions
and methods of using the same of the invention. In some
embodiments, the bacteria is a pathogen. In some embodiments, the
pathogen is a Streptococcus species responsible for strep throat,
meningitis, bacterial pneumonia, endocarditis, erysipelas and/or
necrotizing fasciitis. A variety of Streptococcus species find use
in the compositions and methods of the invention including S.
pneumoniae, S. mutans, S. mitis, S. sanguinis, S. salivarius, S.
viridans, S. salivarius ssp. thermophilus, S. constellatus, S.
pyogenes, S. agalactiae, S. zooepidemicus, Streptococcus bovis and
Streptococcus equines, Streptococcus canis, as well as former Group
D Streptococci including S. faecalis, S. faecium, S. durans, and S.
avium.
[0087] In some preferred embodiments, the bacteria of the genus
Streptococci is S. pneumoniae. In some embodiments, an immunogenic
composition comprising a nanoemulsion and S. pneumoniae antigen may
comprise antigens (e.g., polysaccharide, protein, killed whole
cells (e.g., conjugated or non-conjugated antigens)), wherein the
antigens are derived from multiple (e.g., at least 2, 3, 5, 7, 10,
15, 20, 30, 40, 50, 60, 70, 80 or more) serotypes of S. pneumoniae.
The number of S. pneumoniae antigens utilized can range from 10
different serotypes to about 20 different serotypes. In another
embodiment of the invention, the vaccine may comprise conjugated S.
pneumoniae saccharides and unconjugated S. pneumoniae saccharides.
For example, the invention may comprise 10 conjugated serotypes and
10 unconjugated saccharides. In some embodiments, an immunogenic
composition comprising a nanoemulsion and S. pneumoniae antigen may
comprise S. pneumoniae antigen (e.g., whole cell, polysaccharide,
protein, etc.) from every known and/or isolated serotype.
[0088] In some embodiments, an immunogenic composition comprising a
nanoemulsion and S. pneumoniae antigen comprises S. pneumoniae
antigen (e.g., polysaccharide, protein, killed whole cells)
selected from the following serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8,
9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F
and 33F, although it is appreciated other serotypes could be
substituted depending on the age of the recipient receiving the
vaccine and the geographical location where the vaccine will be
administered. In some embodiments, a 10-valent vaccine refers to a
composition comprising a nanoemulsion and S. pneumoniae antigen,
wherein the S. pneumoniae antigen comprises antigen (e.g.,
polysaccharide, protein, killed whole cells) from 10 S. pneumoniae
serotypes (e.g., serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and
23F). In some embodiments, a 1-valent vaccine refers to a
composition comprising a nanoemulsion and S. pneumoniae antigen,
wherein the S. pneumoniae antigen comprises antigen (e.g.,
polysaccharide, protein, killed whole cells) from one S. pneumoniae
serotype 3. In some embodiments, certain immunogenic compositions
comprising a nanoemulsion and S. pneumoniae antigen comprise S.
pneumoniae antigen (e.g., polysaccharide, protein, killed whole
cells) from a variety of serotypes associated with pediatric
infection (e.g., may comprise serotypes 6A and 19A, or 6A and 22F,
or 19A and 22F, or 6A and 15B, or 19A and 15B, or 22F and 15B). In
some embodiments, certain immunogenic compositions comprising a
nanoemulsion and S. pneumoniae antigen comprise S. pneumoniae
antigen (e.g., polysaccharide, protein, killed whole cells) from a
variety of serotypes associated with infection of the elderly
(e.g., may comprise the serotypes 6A and 19A, or 6A and 22F, or 19A
and 22F, or 6A and 15B, or 19A and 15B, or 22F and 15B,
supplemented with serotypes 19A and 22F, 8 and 12F, or 8 and 15B,
or 8 and 19A, or 8 and 22F, or 12F and 15B, or 12F and 19A, or 12F
and 22F, or 15B and 19A, or 15B and 22F).
[0089] In some embodiments, certain immunogenic compositions
comprising a nanoemulsion and S. pneumoniae antigen comprise S.
pneumoniae antigen (e.g., polysaccharide, protein, killed whole
cells) from a variety of serotypes comprising serotypes 1, 2, 3, 4,
5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F,
20, 22F, 23F and 33F.
[0090] In some embodiments, an immunogenic composition comprising a
nanoemulsion and Streptococcus (e.g., Streptococcus pneumoniae)
antigen comprises protein D (PD) from Haemophilus influenzae (see
e.g. EP 0594610). Haemophilus influenzae is a key causative
organism of otitis media (e.g., thereby protecting against
Haemophilus influenzae related otitis media). In one embodiment,
the vaccine composition comprises protein D. In one aspect, PD is
present as a carrier protein. In another aspect, protein D is
present in the vaccine composition as a free protein. In a further
aspect, protein D is present both as a carrier protein and as free
protein. Protein D may be used as a full length protein or as a
fragment (See, e.g., WO0056360).
[0091] In some embodiments, an immunogenic composition comprising a
nanoemulsion and Streptococcus (e.g., Streptococcus pneumoniae)
antigen comprises one, two or more different types of carrier
protein (e.g., that act as carriers for proteins, saccharides,
etc.). For example, in one embodiment, two or more different
saccharides or proteins may be conjugated to the same carrier
protein, either to the same molecule of carrier protein or to
different molecules of the same carrier protein. Carrier proteins
may be TT, DT, CRM197, fragment C of TT, PhtD, PhtBE or PhtDE
fusions (particularly those described in WO 01/98334 and WO
03/54007), detoxified pneumolysin and protein D. In some
embodiments, a carrier protein present in a composition comprising
a nanoemulsion and Streptococcus (e.g., Streptococcus pneumoniae)
antigen is a member of the polyhistidine triad family (Pht)
proteins, fragments or fusion proteins thereof. The PhtA, PhtB,
PhtD or PhtE proteins may have an amino acid sequence sharing 80%,
85%, 90%, 95%, 98%, 99% or 100% identity with a sequence disclosed
in WO 00/37105 or WO 00/39299 (e.g. with amino acid sequence 1-838
or 21-838 of SEQ ID NO: 4 of WO 00/37105 for PhtD). For example,
fusion proteins are composed of full length or fragments of 2, 3 or
4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins are
PhtA/B, PhtA/D, PhtA/E, PhtB/A, PhtB/D, PhtB/E. PhtD/A. PhtD/B,
PhtD/E, PhtE/A, PhtE/B and PhtE/D, wherein the proteins are linked
with the first mentioned at the N-terminus (see for example
WO01/98334). Carriers may comprise histidine triad motif(s) and/or
coiled coil regions. A histidine triad motif is the portion of
polypeptide that has the sequence HxxHxH where H is histidine and x
is an amino acid other than histidine. A coiled coil region is a
region predicted by "Coils" algorithm Lupus, A et al (1991) Science
252; 1162-1164.
[0092] Examples of carrier proteins which may be used in the
present invention are DT (Diphtheria toxoid), TT (tetanus toxoid)
or fragment C of TT, DT CRM197 (a DT mutant) other DT point
mutants, such as CRM176, CRM228, CRM 45 (Uchida et al J. Biol.
Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and
CRM107 and other mutations described by Nicholls and Youle in
Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc,
1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala
158 to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017
or U.S. Pat. No. 4,950,740; mutation of at least one or more
residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other
mutations disclosed in U.S. Pat. No. 5,917,017 or U.S. Pat. No.
6,455,673; or fragment disclosed in U.S. Pat. No. 5,843,711,
pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63;
2706-13) including ply detoxified in some fashion for example
dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX,
including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins for
example PhtDE fusions, PhtBE fusions (WO 01/98334 and WO 03/54007),
(Pht A-E are described in more detail below) OMPC (meningococcal
outer membrane protein--usually extracted from N. meningitidis
serogroup B--EP0372501), PorB (from N. meningitidis), PD
(Haemophilus influenzae protein D--see, e.g., EP 0 594 610 B), or
immunologically functional equivalents thereof, synthetic peptides
(EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO
94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines,
lymphokines, growth factors or hormones (WO 91/01146), artificial
proteins comprising multiple human CD4+ T cell epitopes from
various pathogen derived antigens (Falugi et al (2001) Eur J
Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al (2004)
Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO
02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C.
difficile (WO 00/61761).
Generation of Antibodies
[0093] An immunogenic composition comprising a nanoemulsion and
Streptococcus (e.g., Streptococcus pneumoniae) antigen can be used
to immunize a mammal, such as a mouse, rat, rabbit, guinea pig,
monkey, or human, to produce polyclonal antibodies. If desired, a
Streptococcus (e.g., Streptococcus pneumoniae) antigen can be
conjugated to a carrier protein, such as bovine serum albumin,
thyroglobulin, keyhole limpet hemocyanin or other carrier described
herein. Depending on the host species, various adjuvants can be
used to increase the immunological response. Such adjuvants
include, but are not limited to, Freund's adjuvant, mineral gels
(e.g., aluminum hydroxide), and surface active substances (e.g.
lysolecithin, pluronic polyols, polyanions, peptides, nanoemulsions
described herein, keyhole limpet hemocyanin, and dinitrophenol).
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially useful.
[0094] Monoclonal antibodies that specifically bind to a
Streptococcus (e.g., Streptococcus pneumoniae) antigen can be
prepared using any technique which provides for the production of
antibody molecules by continuous cell lines in culture. These
techniques include, but are not limited to, the hybridoma
technique, the human B cell hybridoma technique, and the EBV
hybridoma technique (See, e.g., Kohler et al., Nature 256, 495 497,
1985; Kozbor et al., J. Immunol. Methods 81, 3142, 1985; Cote et
al., Proc. Natl. Acad. Sci. 80, 2026 2030, 1983; Cole et al., Mol.
Cell. Biol. 62, 109 120, 1984).
[0095] In addition, techniques developed for the production of
"chimeric antibodies," the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity, can be used (See, e.g.,
Morrison et al., Proc. Natl. Acad. Sci. 81, 68516855, 1984;
Neuberger et al., Nature 312, 604 608, 1984; Takeda et al., Nature
314, 452 454, 1985). Monoclonal and other antibodies also can be
"humanized" to prevent a patient from mounting an immune response
against the antibody when it is used therapeutically. Such
antibodies may be sufficiently similar in sequence to human
antibodies to be used directly in therapy or may require alteration
of a few key residues. Sequence differences between rodent
antibodies and human sequences can be minimized by replacing
residues which differ from those in the human sequences by site
directed mutagenesis of individual residues or by grating of entire
complementarity determining regions.
[0096] Alternatively, humanized antibodies can be produced using
recombinant methods, as described below. Antibodies which
specifically bind to a particular antigen can contain antigen
binding sites which are either partially or fully humanized, as
disclosed in U.S. Pat. No. 5,565,332.
[0097] Alternatively, techniques described for the production of
single chain antibodies can be adapted using methods known in the
art to produce single chain antibodies which specifically bind to a
particular antigen. Antibodies with related specificity, but of
distinct idiotypic composition, can be generated by chain shuffling
from random combinatorial immunoglobin libraries (See, e.g.,
Burton, Proc. Natl. Acad. Sci. 88, 11120 23, 1991).
[0098] Single-chain antibodies also can be constructed using a DNA
amplification method, such as PCR, using hybridoma cDNA as a
template (See, e.g., Thirion et al., 1996, Eur. J. Cancer Prev. 5,
507-11). Single-chain antibodies can be mono- or bispecific, and
can be bivalent or tetravalent. Construction of tetravalent,
bispecific single-chain antibodies is taught, for example, in
Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63.
Construction of bivalent, bispecific single-chain antibodies is
taught, for example, in Mallender & Voss, 1994, J. Biol. Chem.
269, 199-206.
[0099] A nucleotide sequence encoding a single-chain antibody can
be constructed using manual or automated nucleotide synthesis,
cloned into an expression construct using standard recombinant DNA
methods, and introduced into a cell to express the coding sequence,
as described below. Alternatively, single-chain antibodies can be
produced directly using, for example, filamentous phage technology
(See, e.g., Verhaar et al., 1995, Int. J. Cancer 61, 497-501;
Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91).
[0100] Antibodies which specifically bind to a particular antigen
also can be produced by inducing in vivo production in the
lymphocyte population or by screening immunoglobulin libraries or
panels of highly specific binding reagents as disclosed in the
literature (See, e.g., Orlandi et al., Proc. Natl. Acad. Sci. 86,
3833 3837, 1989; Winter et al., Nature 349, 293 299, 1991).
[0101] Chimeric antibodies can be constructed as disclosed in WO
93/03151. Binding proteins which are derived from immunoglobulins
and which are multivalent and multispecific, such as the
"diabodies" described in WO 94/13804, also can be prepared.
Antibodies can be purified by methods well known in the art. For
example, antibodies can be affinity purified by passage over a
column to which the relevant antigen is bound. The bound antibodies
can then be eluted from the column using a buffer with a high salt
concentration.
Nanoemulsions
[0102] The present invention is not limited by the type of
nanoemulsion adjuvant utilized (e.g., for respiratory
administration). Indeed, a variety of nanoemulsion adjuvants are
contemplated to be useful in the present invention.
[0103] For example, in some embodiments, a nanoemulsion comprises
(i) an aqueous phase; (ii) an oil phase; and at least one
additional compound. In some embodiments of the present invention,
these additional compounds are admixed into either the aqueous or
oil phases of the composition. In other embodiments, these
additional compounds are admixed into a composition of previously
emulsified oil and aqueous phases. In certain of these embodiments,
one or more additional compounds are admixed into an existing
emulsion composition immediately prior to its use. In other
embodiments, one or more additional compounds are admixed into an
existing emulsion composition prior to the compositions immediate
use.
[0104] Additional compounds suitable for use in a nanoemulsion of
the present invention include, but are not limited to, one or more
organic, and more particularly, organic phosphate based solvents,
surfactants and detergents, cationic halogen containing compounds,
germination enhancers, interaction enhancers, food additives (e.g.,
flavorings, sweeteners, bulking agents, and the like) and
pharmaceutically acceptable compounds (e.g., carriers). Certain
exemplary embodiments of the various compounds contemplated for use
in the compositions of the present invention are presented below.
Unless described otherwise, nanoemulsions are described in
undiluted form.
[0105] Nanoemulsion adjuvant compositions of the present invention
are not limited to any particular nanoemulsion. Any number of
suitable nanoemulsion compositions may be utilized in the vaccine
compositions of the present invention, including, but not limited
to, those disclosed in Hamouda et al., J. Infect Dis., 180:1939
(1999); Hamouda and Baker, J. Appl. Microbiol., 89:397 (2000); and
Donovan et al., Antivir. Chem. Chemother., 11:41 (2000). Preferred
nanoemulsions of the present invention are those that are non-toxic
to animals. In preferred embodiments, nanoemulsions utilized in the
methods of the present invention are stable, and do not decompose
even after long storage periods (e.g., one or more years).
Additionally, preferred emulsions maintain stability even after
exposure to high temperature and freezing. This is especially
useful if they are to be applied in extreme conditions (e.g.,
extreme heat or cold).
[0106] Some embodiments of the present invention employ an oil
phase containing ethanol. For example, in some embodiments, the
emulsions of the present invention contain (i) an aqueous phase and
(ii) an oil phase containing ethanol as the organic solvent and
optionally a germination enhancer, and (iii) TYLOXAPOL as the
surfactant (preferably 2-5%, more preferably 3%). This formulation
is highly efficacious for inactivation of pathogens and is also
non-irritating and non-toxic to mammalian subjects (e.g., and thus
can be used for administration to a mucosal surface).
[0107] In some other embodiments, the emulsions of the present
invention comprise a first emulsion emulsified within a second
emulsion, wherein (a) the first emulsion comprises (i) an aqueous
phase; and (ii) an oil phase comprising an oil and an organic
solvent; and (iii) a surfactant; and (b) the second emulsion
comprises (i) an aqueous phase; and (ii) an oil phase comprising an
oil and a cationic containing compound; and (iii) a surfactant.
Exemplary Formulations
[0108] The following description provides a number of exemplary
emulsions including formulations for compositions BCTP and
X.sub.8W.sub.60PC. BCTP comprises a water-in oil nanoemulsion, in
which the oil phase was made from soybean oil, tri-n-butyl
phosphate, and TRITON X-100 in 80% water. X.sub.8W.sub.60PC
comprises a mixture of equal volumes of BCTP with W.sub.808P.
W.sub.808P is a liposome-like compound made of glycerol
monostearate, refined oya sterols (e.g., GENEROL sterols), TWEEN
60, soybean oil, a cationic ion halogen-containing CPC and
peppermint oil. The GENEROL family are a group of a polyethoxylated
soya sterols (Henkel Corporation, Ambler, Pa.). Exemplary emulsion
formulations useful in the present invention are provided in Table
1. These particular formulations may be found in U.S. Pat. No.
5,700,679 (NN); U.S. Pat. Nos. 5,618,840; 5,549,901 (W.sub.808P);
and U.S. Pat. No. 5,547,677, each of which is hereby incorporated
by reference in their entireties. Certain other emulsion
formulations are presented U.S. patent application Ser. No.
10/669,865, hereby incorporated by reference in its entirety.
[0109] The X.sub.8W.sub.60PC emulsion is manufactured by first
making the W.sub.808P emulsion and BCTP emulsions separately. A
mixture of these two emulsions is then re-emulsified to produce a
fresh emulsion composition termed X.sub.8W.sub.60PC. Methods of
producing such emulsions are described in U.S. Pat. Nos. 5,103,497
and 4,895,452 (each of which is herein incorporated by reference in
their entireties).
TABLE-US-00001 TABLE 1 Water to Oil Phase Ratio Oil Phase Formula
(Vol/Vol) BCTP 1 vol. Tri(N-butyl)phosphate 4:1 1 vol. TRITON X-100
8 vol. Soybean oil NN 86.5 g Glycerol monooleate 3:1 60.1 ml
Nonoxynol-9 24.2 g GENEROL 122 3.27 g Cetylpyridinium chloride 554
g Soybean oil W.sub.808P 86.5 g Glycerol monooleate 3.2:1 21.2 g
Polysorbate 60 24.2 g GENEROL 122 3.27 g Cetylpyddinium chloride 4
ml Peppermint oil 554 g Soybean oil SS 86.5 g Glycerol monooleate
3.2:1 21.2 g Polysorbate 60 (1% bismuth in water) 24.2 g GENEROL
122 3.27 g Cetylpyridinium chloride 554 g Soybean oil
[0110] The compositions listed above are only exemplary and those
of skill in the art will be able to alter the amounts of the
components to arrive at a nanoemulsion composition suitable for the
purposes of the present invention. Those skilled in the art will
understand that the ratio of oil phase to water as well as the
individual oil carrier, surfactant CPC and organic phosphate
buffer, components of each composition may vary.
[0111] Although certain compositions comprising BCTP have a water
to oil ratio of 4:1, it is understood that the BCTP may be
formulated to have more or less of a water phase. For example, in
some embodiments, there is 3, 4, 5, 6, 7, 8, 9, 10, or more parts
of the water phase to each part of the oil phase. The same holds
true for the W.sub.808P formulation. Similarly, the ratio of
Tri(N-butyl) phosphate: TRITON X-100: soybean oil also may be
varied.
[0112] Although Table 1 lists specific amounts of glycerol
monooleate, polysorbate 60, GENEROL 122, cetylpyridinium chloride,
and carrier oil for W.sub.808P, these are merely exemplary. An
emulsion that has the properties of W.sub.808P may be formulated
that has different concentrations of each of these components or
indeed different components that will fulfill the same function.
For example, the emulsion may have between about 80 to about 100 g
of glycerol monooleate in the initial oil phase. In other
embodiments, the emulsion may have between about 15 to about 30 g
polysorbate 60 in the initial oil phase. In yet another embodiment
the composition may comprise between about 20 to about 30 g of a
GENEROL sterol, in the initial oil phase.
[0113] Individual components of nanoemulsions (e.g. in an
immunogenic composition of the present invention) can function both
to inactivate a pathogen as well as to contribute to the
non-toxicity of the emulsions. For example, the active component in
BCTP, TRITON-X100, shows less ability to inactivate a virus at
concentrations equivalent to 11% BCTP. Adding the oil phase to the
detergent and solvent markedly reduces the toxicity of these agents
in tissue culture at the same concentrations. While not being bound
to any theory (an understanding of the mechanism is not necessary
to practice the present invention, and the present invention is not
limited to any particular mechanism), it is suggested that the
nanoemulsion enhances the interaction of its components with the
pathogens thereby facilitating the inactivation of the pathogen and
reducing the toxicity of the individual components. Furthermore,
when all the components of BCTP are combined in one composition but
are not in a nanoemulsion structure, the mixture is not as
effective at inactivating a pathogen as when the components are in
a nanoemulsion structure.
[0114] Numerous additional embodiments presented in classes of
formulations with like compositions are presented below. The
following compositions recite various ratios and mixtures of active
components. One skilled in the art will appreciate that the below
recited formulation are exemplary and that additional formulations
comprising similar percent ranges of the recited components are
within the scope of the present invention.
[0115] In certain embodiments of the present invention, a
nanoemulsion comprises from about 3 to 8 vol. % of TYLOXAPOL, about
8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride
(CPC), about 60 to 70 vol. % oil (e.g., soybean oil), about 15 to
25 vol. % of aqueous phase (e.g., DiH.sub.2O or PBS), and in some
formulations less than about 1 vol. % of 1N NaOH. Some of these
embodiments comprise PBS. It is contemplated that the addition of
1N NaOH and/or PBS in some of these embodiments, allows the user to
advantageously control the pH of the formulations, such that pH
ranges from about 7.0 to about 9.0, and more preferably from about
7.1 to 8.5 are achieved. For example, one embodiment of the present
invention comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of
ethanol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and
about 24 vol. % of DiH.sub.2O (designated herein as Y3EC). Another
similar embodiment comprises about 3.5 vol. % of TYLOXAPOL, about 8
vol. % of ethanol, and about 1 vol. % of CPC, about 64 vol. % of
soybean oil, and about 23.5 vol. % of DiH.sub.2O (designated herein
as Y3.5EC). Yet another embodiment comprises about 3 vol. % of
TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about
0.067 vol. % of 1N NaOH, such that the pH of the formulation is
about 7.1, about 64 vol. % of soybean oil, and about 23.93 vol. %
of DiH.sub.2O (designated herein as Y3EC pH 7.1). Still another
embodiment comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of
ethanol, about 1 vol. % of CPC, about 0.67 vol. % of 1N NaOH, such
that the pH of the formulation is about 8.5, and about 64 vol. % of
soybean oil, and about 23.33 vol. % of DiH.sub.2O (designated
herein as Y3EC pH 8.5). Another similar embodiment comprises about
4% TYLOXAPOL, about 8 vol. % ethanol, about 1% CPC, and about 64
vol. % of soybean oil, and about 23 vol. % of DiH.sub.2O
(designated herein as Y4EC). In still another embodiment the
formulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1
vol. % of CPC, and about 64 vol. % of soybean oil, and about 19
vol. % of DiH.sub.2O (designated herein as Y8EC). A further
embodiment comprises about 8 vol. % of TYLOXAPOL, about 8 vol. % of
ethanol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and
about 19 vol. % of 1.times.PBS (designated herein as Y8EC PBS).
[0116] In some embodiments of the present invention, a nanoemulsion
comprises about 8 vol. % of ethanol, and about 1 vol. % of CPC, and
about 64 vol. % of oil (e.g., soybean oil), and about 27 vol. % of
aqueous phase (e.g., DiH.sub.2O or PBS) (designated herein as
EC).
[0117] In some embodiments, a nanoemulsion comprises from about 8
vol. % of sodium dodecyl sulfate (SDS), about 8 vol. % of tributyl
phosphate (TBP), and about 64 vol. % of oil (e.g., soybean oil),
and about 20 vol. % of aqueous phase (e.g., DiH.sub.2O or PBS)
(designated herein as S8P).
[0118] In some embodiments, a nanoemulsion comprises from about 1
to 2 vol. % of TRITON X-100, from about 1 to 2 vol. % of TYLOXAPOL,
from about 7 to 8 vol. % of ethanol, about 1 vol. % of
cetylpyridinium chloride (CPC), about 64 to 57.6 vol. % of oil
(e.g., soybean oil), and about 23 vol. % of aqueous phase (e.g.,
DiH.sub.2O or PBS). Additionally, some of these formulations
further comprise about 5 mM of L-alanine/Inosine, and about 10 mM
ammonium chloride. Some of these formulations comprise PBS. It is
contemplated that the addition of PBS in some of these embodiments,
allows the user to advantageously control the pH of the
formulations. For example, one embodiment of the present invention
comprises about 2 vol. % of TRITON X-100, about 2 vol. % of
TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % CPC, about 64
vol. % of soybean oil, and about 23 vol. % of aqueous phase
DiH.sub.2O. In another embodiment the formulation comprises about
1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about
7.2 vol. % of ethanol, about 0.9 vol. % of CPC, about 5 mM
L-alanine/Inosine, and about 10 mM ammonium chloride, about 57.6
vol. % of soybean oil, and the remainder of 1.times.PBS (designated
herein as 90% X2Y2EC/GE).
[0119] In alternative embodiments, a nanoemulsion comprises from
about 5 vol. % of TWEEN 80, from about 8 vol. % of ethanol, from
about 1 vol. % of CPC, about 64 vol. % of oil (e.g., soybean oil),
and about 22 vol. % of DiH.sub.2O (designated herein as
W.sub.805EC). In yet another alternative embodiment, a nanoemulsion
comprises from about 5 vol. % of TWEEN 80, from about 8 vol. % of
ethanol, about 64 vol. % of oil (e.g., soybean oil), and about 23
vol. % of DiH.sub.2O (designated herein as W.sub.805E).
[0120] In some embodiments, the present invention provides a
nanoemulsion comprising from about 5 vol. % of Poloxamer-407, from
about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64
vol. % of oil (e.g., soybean oil), and about 22 vol. % of
DiH.sub.2O (designated herein as P.sub.4075EC). Although an
understanding of the mechanism is not necessary to practice the
present invention, and the present invention is not limited to any
particular mechanism, in some embodiments, a nanoemulsion
comprising Poloxamer-407 does not elicit and/or augment immune
responses (e.g., in the lung) in a subject. In some embodiments,
various dilutions of a nanoemulsion provided herein (e.g.,
P.sub.4075EC) can be utilized to treat (e.g., kill and/or inhibit
growth of) bacteria. In some embodiments, undiluted nanoemulsion is
utilized. In some embodiments, P.sub.4075EC is diluted (e.g., in
serial, two fold dilutions) to obtain a desired concentration of
one of the constituents of the nanoemulsion (e.g., CPC).
[0121] In still other embodiments of the present invention, a
nanoemulsion comprises from about 5 vol. % of TWEEN 20, from about
8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol. % of
oil (e.g., soybean oil), and about 22 vol. % of DiH.sub.2O
(designated herein as W.sub.205EC).
[0122] In still other embodiments of the present invention, a
nanoemulsion comprises from about 2 to 8 vol. % of TRITON X-100,
about 8 vol. % of ethanol, about 1 vol. % of CPC, about 60 to 70
vol. % of oil (e.g., soybean, or olive oil), and about 15 to 25
vol. % of aqueous phase (e.g., DiH.sub.2O or PBS). For example, the
present invention contemplates formulations comprising about 2 vol.
% of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of
soybean oil, and about 26 vol. % of DiH.sub.2O (designated herein
as X2E). In other similar embodiments, a nanoemulsion comprises
about 3 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64
vol. % of soybean oil, and about 25 vol. % of DiH.sub.2O
(designated herein as X3E). In still further embodiments, the
formulations comprise about 4 vol. % Triton of X-100, about 8 vol.
% of ethanol, about 64 vol. % of soybean oil, and about 24 vol. %
of DiH.sub.2O (designated herein as X4E). In yet other embodiments,
a nanoemulsion comprises about 5 vol. % of TRITON X-100, about 8
vol. % of ethanol, about 64 vol. % of soybean oil, and about 23
vol. % of DiH.sub.2O (designated herein as X5E). In some
embodiments, a nanoemulsion comprises about 6 vol. % of TRITON
X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil,
and about 22 vol. % of DiH.sub.2O (designated herein as X6E). In
still further embodiments of the present invention, a nanoemulsion
comprises about 8 vol. % of TRITON X-100, about 8 vol. % of
ethanol, about 64 vol. % of soybean oil, and about 20 vol. % of
DiH.sub.2O (designated herein as X8E). In still further
embodiments, a nanoemulsion comprises about 8 vol. % of TRITON
X-100, about 8 vol. % of ethanol, about 64 vol. % of olive oil, and
about 20 vol. % of DiH.sub.2O (designated herein as X8E 0). In yet
another embodiment, a nanoemulsion comprises 8 vol. % of TRITON
X-100, about 8 vol. % ethanol, about 1 vol. % CPC, about 64 vol. %
of soybean oil, and about 19 vol. % of DiH.sub.2O (designated
herein as X8EC).
[0123] In alternative embodiments of the present invention, a
nanoemulsion comprises from about 1 to 2 vol. % of TRITON X-100,
from about 1 to 2 vol. % of TYLOXAPOL, from about 6 to 8 vol. %
TBP, from about 0.5 to 1.0 vol. % of CPC, from about 60 to 70 vol.
% of oil (e.g., soybean), and about 1 to 35 vol. % of aqueous phase
(e.g., DiH.sub.2O or PBS). Additionally, certain of these
nanoemulsions may comprise from about 1 to 5 vol. % of trypticase
soy broth, from about 0.5 to 1.5 vol. % of yeast extract, about 5
mM L-alanine/Inosine, about 10 mM ammonium chloride, and from about
20-40 vol. % of liquid baby formula. In some embodiments comprising
liquid baby formula, the formula comprises a casein hydrolysate
(e.g., Neutramigen, or Progestimil, and the like). In some of these
embodiments, a nanoemulsion further comprises from about 0.1 to 1.0
vol. % of sodium thiosulfate, and from about 0.1 to 1.0 vol. % of
sodium citrate. Other similar embodiments comprising these basic
components employ phosphate buffered saline (PBS) as the aqueous
phase. For example, one embodiment comprises about 2 vol. % of
TRITON X-100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1
vol. % of CPC, about 64 vol. % of soybean oil, and about 23 vol. %
of DiH.sub.2O (designated herein as X2Y2EC). In still other
embodiments, the inventive formulation comprises about 2 vol. % of
TRITON X-100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1
vol. % of CPC, about 0.9 vol. % of sodium thiosulfate, about 0.1
vol. % of sodium citrate, about 64 vol. % of soybean oil, and about
22 vol. % of DiH.sub.2O (designated herein as X2Y2PC STS1). In
another similar embodiment, a nanoemulsion comprises about 1.7 vol.
% TRITON X-100, about 1.7 vol. % TYLOXAPOL, about 6.8 vol. % TBP,
about 0.85% CPC, about 29.2% NEUTRAMIGEN, about 54.4 vol. % of
soybean oil, and about 4.9 vol. % of DiH.sub.2O (designated herein
as 85% X2Y2PC/baby). In yet another embodiment of the present
invention, a nanoemulsion comprises about 1.8 vol. % of TRITON
X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % of TBP,
about 0.9 vol. % of CPC, about 5 mM L-alanine/Inosine, about 10 mM
ammonium chloride, about 57.6 vol. % of soybean oil, and the
remainder vol. % of 0.1.times.PBS (designated herein as 90% X2Y2
PC/GE). In still another embodiment, a nanoemulsion comprises about
1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about
7.2 vol. % TBP, about 0.9 vol. % of CPC, and about 3 vol. %
trypticase soy broth, about 57.6 vol. % of soybean oil, and about
27.7 vol. % of DiH.sub.2O (designated herein as 90% X2Y2PC/TSB). In
another embodiment of the present invention, a nanoemulsion
comprises about 1.8 vol. % TRITON X-100, about 1.8 vol. %
TYLOXAPOL, about 7.2 vol. % TBP, about 0.9 vol. % CPC, about 1 vol.
% yeast extract, about 57.6 vol. % of soybean oil, and about 29.7
vol. % of DiH.sub.2O (designated herein as 90% X2Y2PC/YE).
[0124] In some embodiments of the present invention, a nanoemulsion
comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and
about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g., soybean
or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g.,
DiH.sub.2O or PBS). In a particular embodiment of the present
invention, a nanoemulsion comprises about 3 vol. % of TYLOXAPOL,
about 8 vol. % of TBP, and about 1 vol. % of CPC, about 64 vol. %
of soybean, and about 24 vol. % of DiH.sub.2O (designated herein as
Y3PC).
[0125] In some embodiments of the present invention, a nanoemulsion
comprises from about 4 to 8 vol. % of TRITON X-100, from about 5 to
8 vol. % of TBP, about 30 to 70 vol. % of oil (e.g., soybean or
olive oil), and about 0 to 30 vol. % of aqueous phase (e.g.,
DiH.sub.2O or PBS). Additionally, certain of these embodiments
further comprise about 1 vol. % of CPC, about 1 vol. % of
benzalkonium chloride, about 1 vol. % cetylyridinium bromide, about
1 vol. % cetyldimethyletylammonium bromide, 500 .mu.M EDTA, about
10 mM ammonium chloride, about 5 mM Inosine, and about 5 mM
L-alanine. For example, in a certain preferred embodiment, a
nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol.
% of TBP, about 64 vol. % of soybean oil, and about 20 vol. % of
DiH.sub.2O (designated herein as X8P). In another embodiment of the
present invention, a nanoemulsion comprises about 8 vol. % of
TRITON X-100, about 8 vol. % of TBP, about 1% of CPC, about 64 vol.
% of soybean oil, and about 19 vol. % of DiH.sub.2O (designated
herein as X8PC). In still another embodiment, a nanoemulsion
comprises about 8 vol. % TRITON X-100, about 8 vol. % of TBP, about
1 vol. % of CPC, about 50 vol. % of soybean oil, and about 33 vol.
% of DiH.sub.2O (designated herein as ATB-X1001). In yet another
embodiment, the formulations comprise about 8 vol. % of TRITON
X-100, about 8 vol. % of TBP, about 2 vol. % of CPC, about 50 vol.
% of soybean oil, and about 32 vol. % of DiH.sub.2O (designated
herein as ATB-X002). In some embodiments, a nanoemulsion comprises
about 4 vol. % TRITON X-100, about 4 vol. % of TBP, about 0.5 vol.
% of CPC, about 32 vol. % of soybean oil, and about 59.5 vol. % of
DiH.sub.2O (designated herein as 50% X8PC). In some embodiments, a
nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol.
% of TBP, about 0.5 vol. % CPC, about 64 vol. % of soybean oil, and
about 19.5 vol. % of DiH.sub.2O (designated herein as
X8PC.sub.1/2). In some embodiments of the present invention, a
nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol.
% of TBP, about 2 vol. % of CPC, about 64 vol. % of soybean oil,
and about 18 vol. % of DiH.sub.2O (designated herein as X8PC2). In
other embodiments, a nanoemulsion comprises about 8 vol. % of
TRITON X-100, about 8% of TBP, about 1% of benzalkonium chloride,
about 50 vol. % of soybean oil, and about 33 vol. % of DiH.sub.2O
(designated herein as X8P BC). In an alternative embodiment of the
present invention, a nanoemulsion comprises about 8 vol. % of
TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of
cetylyridinium bromide, about 50 vol. % of soybean oil, and about
33 vol. % of DiH.sub.2O (designated herein as X8P CPB). In another
exemplary embodiment of the present invention, a nanoemulsion
comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP,
about 1 vol. % of cetyldimethyletylammonium bromide, about 50 vol.
% of soybean oil, and about 33 vol. % of DiH.sub.2O (designated
herein as X8P CTAB). In still further embodiments, a nanoemulsion
comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP,
about 1 vol. % of CPC, about 500 .mu.M EDTA, about 64 vol. % of
soybean oil, and about 15.8 vol. % DiH.sub.2O (designated herein as
X8PC EDTA). In some embodiments, a nanoemulsion comprises 8 vol. %
of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC,
about 10 mM ammonium chloride, about 5 mM Inosine, about 5 mM
L-alanine, about 64 vol. % of soybean oil, and about 19 vol. % of
DiH.sub.2O or PBS (designated herein as X8PC GE.sub.1x). In another
embodiment of the present invention, a nanoemulsion comprises about
5 vol. % of TRITON X-100, about 5% of TBP, about 1 vol. % of CPC,
about 40 vol. % of soybean oil, and about 49 vol. % of DiH.sub.2O
(designated herein as X5P.sub.5C).
[0126] In some embodiments of the present invention, a nanoemulsion
comprises about 2 vol. % TRITON X-100, about 6 vol. % TYLOXAPOL,
about 8 vol. % ethanol, about 64 vol. % of soybean oil, and about
20 vol. % of DiH.sub.2O (designated herein as X2Y6E).
[0127] In an additional embodiment of the present invention, a
nanoemulsion comprises about 8 vol. % of TRITON X-100, and about 8
vol. % of glycerol, about 60 to 70 vol. % of oil (e.g., soybean or
olive oil), and about 15 to 25 vol. % of aqueous phase (e.g.,
DiH.sub.2O or PBS). Certain nanoemulsion compositions (e.g., used
to generate an immune response (e.g., for use as a vaccine)
comprise about 1 vol. % L-ascorbic acid. For example, one
particular embodiment comprises about 8 vol. % of TRITON X-100,
about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and
about 20 vol. % of DiH.sub.2O (designated herein as X8G). In still
another embodiment, a nanoemulsion comprises about 8 vol. % of
TRITON X-100, about 8 vol. % of glycerol, about 1 vol. % of
L-ascorbic acid, about 64 vol. % of soybean oil, and about 19 vol.
% of DiH.sub.2O (designated herein as X8GV.sub.c).
[0128] In still further embodiments, a nanoemulsion comprises about
8 vol. % of TRITON X-100, from about 0.5 to 0.8 vol. % of TWEEN 60,
from about 0.5 to 2.0 vol. % of CPC, about 8 vol. % of TBP, about
60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15
to 25 vol. % of aqueous phase (e.g., DiH.sub.2O or PBS). For
example, in one particular embodiment a nanoemulsion comprises
about 8 vol. % of TRITON X-100, about 0.70 vol. % of TWEEN 60,
about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of
soybean oil, and about 18.3 vol. % of DiH.sub.2O (designated herein
as X8W60PC.sub.1). In some embodiments, a nanoemulsion comprises
about 8 vol. % of TRITON X-100, about 0.71 vol. % of TWEEN 60,
about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of
soybean oil, and about 18.29 vol. % of DiH.sub.2O (designated
herein as W60.sub.0.7X8PC). In yet other embodiments, a
nanoemulsion comprises from about 8 vol. % of TRITON X-100, about
0.7 vol. % of TWEEN 60, about 0.5 vol. % of CPC, about 8 vol. % of
TBP, about 64 to 70 vol. % of soybean oil, and about 18.8 vol. % of
DiH.sub.2O (designated herein as X8W60PC.sub.2). In still other
embodiments, a nanoemulsion comprises about 8 vol. % of TRITON
X-100, about 0.71 vol. % of TWEEN 60, about 2 vol. % of CPC, about
8 vol. % of TBP, about 64 vol. % of soybean oil, and about 17.3
vol. % of DiH.sub.2O. In another embodiment of the present
invention, a nanoemulsion comprises about 0.71 vol. % of TWEEN 60,
about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of
soybean oil, and about 25.29 vol. % of DiH.sub.2O (designated
herein as W60.sub.0.7PC).
[0129] In another embodiment of the present invention, a
nanoemulsion comprises about 2 vol. % of dioctyl sulfosuccinate,
either about 8 vol. % of glycerol, or about 8 vol. % TBP, in
addition to, about 60 to 70 vol. % of oil (e.g., soybean or olive
oil), and about 20 to 30 vol. % of aqueous phase (e.g., DiH.sub.2O
or PBS). For example, in some embodiments, a nanoemulsion comprises
about 2 vol. % of dioctyl sulfosuccinate, about 8 vol. % of
glycerol, about 64 vol. % of soybean oil, and about 26 vol. % of
DiH.sub.2O (designated herein as D2G). In another related
embodiment, a nanoemulsion comprises about 2 vol. % of dioctyl
sulfosuccinate, and about 8 vol. % of TBP, about 64 vol. % of
soybean oil, and about 26 vol. % of DiH.sub.2O (designated herein
as D2P).
[0130] In still other embodiments of the present invention, a
nanoemulsion comprises about 8 to 10 vol. % of glycerol, and about
1 to 10 vol. % of CPC, about 50 to 70 vol. % of oil (e.g., soybean
or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g.,
DiH.sub.2O or PBS). Additionally, in certain of these embodiments,
a nanoemulsion further comprises about 1 vol. % of L-ascorbic acid.
For example, in some embodiments, a nanoemulsion comprises about 8
vol. % of glycerol, about 1 vol. % of CPC, about 64 vol. % of
soybean oil, and about 27 vol. % of DiH.sub.2O (designated herein
as GC). In some embodiments, a nanoemulsion comprises about 10 vol.
% of glycerol, about 10 vol. % of CPC, about 60 vol. % of soybean
oil, and about 20 vol. % of DiH.sub.2O (designated herein as GC10).
In still another embodiment of the present invention, a
nanoemulsion comprises about 10 vol. % of glycerol, about 1 vol. %
of CPC, about 1 vol. % of L-ascorbic acid, about 64 vol. % of
soybean or oil, and about 24 vol. % of DiH.sub.2O (designated
herein as GCV.sub.c).
[0131] In some embodiments of the present invention, a nanoemulsion
comprises about 8 to 10 vol. % of glycerol, about 8 to 10 vol. % of
SDS, about 50 to 70 vol. % of oil (e.g., soybean or olive oil), and
about 15 to 30 vol. % of aqueous phase (e.g., DiH.sub.2O or PBS).
Additionally, in certain of these embodiments, a nanoemulsion
further comprise about 1 vol. % of lecithin, and about 1 vol. % of
p-Hydroxybenzoic acid methyl ester. Exemplary embodiments of such
formulations comprise about 8 vol. % SDS, 8 vol. % of glycerol,
about 64 vol. % of soybean oil, and about 20 vol. % of DiH.sub.2O
(designated herein as S8G). A related formulation comprises about 8
vol. % of glycerol, about 8 vol. % of SDS, about 1 vol. % of
lecithin, about 1 vol. % of p-Hydroxybenzoic acid methyl ester,
about 64 vol. % of soybean oil, and about 18 vol. % of DiH.sub.2O
(designated herein as S8GL1B1).
[0132] In yet another embodiment of the present invention, a
nanoemulsion comprises about 4 vol. % of TWEEN 80, about 4 vol. %
of TYLOXAPOL, about 1 vol. % of CPC, about 8 vol. % of ethanol,
about 64 vol. % of soybean oil, and about 19 vol. % of DiH.sub.2O
(designated herein as W.sub.804 Y4EC).
[0133] In some embodiments of the present invention, a nanoemulsion
comprises about 0.01 vol. % of CPC, about 0.08 vol. % of TYLOXAPOL,
about 10 vol. % of ethanol, about 70 vol. % of soybean oil, and
about 19.91 vol. % of DiH.sub.2O (designated herein as
Y.08EC.01).
[0134] In yet another embodiment of the present invention, a
nanoemulsion comprises about 8 vol. % of sodium lauryl sulfate, and
about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and
about 20 vol. % of DiH.sub.2O (designated herein as SLS8G).
[0135] The specific formulations described above are simply
examples to illustrate the variety of nanoemulsion adjuvants that
find use in the present invention. The present invention
contemplates that many variations of the above formulations, as
well as additional nanoemulsions, find use in the methods of the
present invention. Candidate emulsions can be easily tested to
determine if they are suitable. First, the desired ingredients are
prepared using the methods described herein, to determine if an
emulsion can be formed. If an emulsion cannot be formed, the
candidate is rejected. For example, a candidate composition made of
4.5% sodium thiosulfate, 0.5% sodium citrate, 10% n-butanol, 64%
soybean oil, and 21% DiH.sub.2O does not form an emulsion.
[0136] Second, the candidate emulsion should form a stable
emulsion. An emulsion is stable if it remains in emulsion form for
a sufficient period to allow its intended use (e.g., to generate an
immune response in a subject). For example, for emulsions that are
to be stored, shipped, etc., it may be desired that the composition
remain in emulsion form for months to years. Typical emulsions that
are relatively unstable, will lose their form within a day. For
example, a candidate composition made of 8% 1-butanol, 5% TWEEN 10,
1% CPC, 64% soybean oil, and 22% DiH.sub.2O does not form a stable
emulsion. Nanoemulsions that have been shown to be stable include,
but are not limited to, 8 vol. % of TRITON X-100, about 8 vol. % of
TBP, about 64 vol. % of soybean oil, and about 20 vol. % of
DiH.sub.2O (designated herein as X8P); 5 vol. % of TWEEN 20, from
about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64
vol. % of oil (e.g., soybean oil), and about 22 vol. % of
DiH.sub.2O (designated herein as W.sub.205EC); 0.08% Triton X-100,
0.08% Glycerol, 0.01% Cetylpyridinium Chloride, 99% Butter, and
0.83% diH.sub.2O (designated herein as 1% X8GC Butter); 0.8% Triton
X-100, 0.8% Glycerol, 0.1% Cetylpyridinium Chloride, 6.4% Soybean
Oil, 1.9% diH.sub.2O, and 90% Butter (designated herein as 10% X8GC
Butter); 2% W.sub.205EC, 1% Natrosol 250L NF, and 97% diH.sub.2O
(designated herein as 2% W.sub.205EC L GEL); 1% Cetylpyridinium
Chloride, 5% TWEEN 20, 8% Ethanol, 64% 70 Viscosity Mineral Oil,
and 22% diH.sub.2O (designated herein as W.sub.205EC 70 Mineral
Oil); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% 350
Viscosity Mineral Oil, and 22% diH.sub.2O (designated herein as
W.sub.205EC 350 Mineral Oil). In some embodiments, nanoemulsions of
the present invention are stable for over a week, over a month, or
over a year.
[0137] Third, the candidate emulsion should have efficacy for its
intended use. For example, a nanoemulsion should inactivate (e.g.,
kill or inhibit growth of) a pathogen to a desired level (e.g., 1
log, 2 log, 3 log, 4 log, . . . reduction). Using the methods
described herein, one is capable of determining the suitability of
a particular candidate emulsion against the desired pathogen.
Generally, this involves exposing the pathogen to the emulsion for
one or more time periods in a side-by-side experiment with the
appropriate control samples (e.g., a negative control such as
water) and determining if, and to what degree, the emulsion
inactivates (e.g., kills and/or neutralizes) the microorganism. For
example, a candidate composition made of 1% ammonium chloride, 5%
TWEEN 20, 8% ethanol, 64% soybean oil, and 22% DiH.sub.2O was shown
not to be an effective emulsion. The following candidate emulsions
were shown to be effective using the methods described herein: 5%
TWEEN 20, 5% Cetylpyridinium Chloride, 10% Glycerol, 60% Soybean
Oil, and 20% diH.sub.2O (designated herein as W.sub.205 GC5); 1%
Cetylpyridinium Chloride, 5% TWEEN 20, 10% Glycerol, 64% Soybean
Oil, and 20% diH.sub.2O (designated herein as W.sub.205 GC); 1%
Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% Olive Oil,
and 22% diH.sub.2O (designated herein as W.sub.205EC Olive Oil); 1%
Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% Flaxseed
Oil, and 22% diH.sub.2O (designated herein as W.sub.205EC Flaxseed
Oil); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64%
Corn Oil, and 22% diH.sub.2O (designated herein as W.sub.205EC Corn
Oil); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64%
Coconut Oil, and 22% diH.sub.2O (designated herein as W.sub.205EC
Coconut Oil); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol,
64% Cottonseed Oil, and 22% diH.sub.2O (designated herein as
W.sub.205EC Cottonseed Oil); 8% Dextrose, 5% TWEEN 10, 1%
Cetylpyridinium Chloride, 64% Soybean Oil, and 22% diH.sub.2O
(designated herein as W.sub.205C Dextrose); 8% PEG 200, 5% TWEEN
10, 1% Cetylpyridinium Chloride, 64% Soybean Oil, and 22%
diH.sub.2O (designated herein as W.sub.205C PEG 200); 8% Methanol,
5% TWEEN 10, 1% Cetylpyridinium Chloride, 64% Soybean Oil, and 22%
diH.sub.2O (designated herein as W.sub.205C Methanol); 8% PEG 1000,
5% TWEEN 10, 1% Cetylpyridinium Chloride, 64% Soybean Oil, and 22%
diH.sub.2O (designated herein as W.sub.205C PEG 1000); 2%
W.sub.205EC, 2% Natrosol 250H NF, and 96% diH.sub.2O (designated
herein as 2% W.sub.205EC Natrosol 2, also called 2% W.sub.205EC
GEL); 2% W.sub.205EC, 1% Natrosol 250H NF, and 97% diH.sub.2O
(designated herein as 2% W.sub.205EC Natrosol 1); 2% W.sub.205EC,
3% Natrosol 250H NF, and 95% diH.sub.2O (designated herein as 2%
W.sub.205EC Natrosol 3); 2% W.sub.205EC, 0.5% Natrosol 250H NF, and
97.5% diH.sub.2O (designated herein as 2% W.sub.205EC Natrosol
0.5); 2% W.sub.205EC, 2% Methocel A, and 96% diH.sub.2O (designated
herein as 2% W.sub.205EC Methocel A); 2% W.sub.205EC, 2% Methocel
K, and 96% diH.sub.2O (designated herein as 2% W.sub.205EC Methocel
K); 2% Natrosol, 0.1% X8PC, 0.1.times.PBS, 5 mM L-alanine, 5 mM
Inosine, 10 mM Ammonium Chloride, and diH.sub.2O (designated herein
as 0.1% X8PC/GE+2% Natrosol); 2% Natrosol, 0.8% Triton X-100, 0.8%
Tributyl Phosphate, 6.4% Soybean Oil, 0.1% Cetylpyridinium
Chloride, 0.1.times.PBS, 5 mM L-alanine, 5 mM Inosine, 10 mM
Ammonium Chloride, and diH.sub.2O (designated herein as 10%
X8PC/GE+2% Natrosol); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8%
Ethanol, 64% Lard, and 22% diH.sub.2O (designated herein as
W.sub.205EC Lard); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8%
Ethanol, 64% Mineral Oil, and 22% diH.sub.2O (designated herein as
W.sub.205EC Mineral Oil); 0.1% Cetylpyridinium Chloride, 2%
Nerolidol, 5% TWEEN 20, 10% Ethanol, 64% Soybean Oil, and 18.9%
diH.sub.2O (designated herein as W.sub.205EC.sub.0.1N); 0.1%
Cetylpyridinium Chloride, 2% Farnesol, 5% TWEEN 20, 10% Ethanol,
64% Soybean Oil, and 18.9% diH.sub.2O (designated herein as
W.sub.205EC.sub.0.1F); 0.1% Cetylpyridinium Chloride, 5% TWEEN 20,
10% Ethanol, 64% Soybean Oil, and 20.9% diH.sub.2O (designated
herein as W.sub.205EC.sub.0.1); 10% Cetylpyridinium Chloride, 8%
Tributyl Phosphate, 8% Triton X-100, 54% Soybean Oil, and 20%
diH.sub.2O (designated herein as X8PC.sub.10); 5% Cetylpyridinium
Chloride, 8% Triton X-100, 8% Tributyl Phosphate, 59% Soybean Oil,
and 20% diH.sub.2O (designated herein as X8PC.sub.5); 0.02%
Cetylpyridinium Chloride, 0.1% TWEEN 20, 10% Ethanol, 70% Soybean
Oil, and 19.88% diH.sub.2O (designated herein as W.sub.200.1
EC.sub.0.02); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8%
Glycerol, 64% Mobil 1, and 22% diH.sub.2O (designated herein as
W.sub.205 GC Mobil 1); 7.2% Triton X-100, 7.2% Tributyl Phosphate,
0.9% Cetylpyridinium Chloride, 57.6% Soybean Oil, 0.1.times.PBS, 5
mM L-alanine, 5 mM Inosine, 10 mM Ammonium Chloride, and 25.87%
diH.sub.2O (designated herein as 90% X8PC/GE); 7.2% Triton X-100,
7.2% Tributyl Phosphate, 0.9% Cetylpyridinium Chloride, 57.6%
Soybean Oil, 1% EDTA, 5 mM L-alanine, 5 mM Inosine, 10 mM Ammonium
Chloride, 0.1.times.PBS, and diH.sub.2O (designated herein as 90%
X8PC/GE EDTA); and 7.2% Triton X-100, 7.2% Tributyl Phosphate, 0.9%
Cetylpyridinium Chloride, 57.6% Soybean Oil, 1% Sodium Thiosulfate,
5 mM L-alanine, 5 mM Inosine, 10 mM Ammonium Chloride,
0.1.times.PBS, and diH.sub.2O (designated herein as 90% X8PC/GE
STS).
[0138] In preferred embodiments of the present invention, the
nanoemulsions are non-toxic (e.g., to humans, plants, or animals),
non-irritant (e.g., to humans, plants, or animals), and
non-corrosive (e.g., to humans, plants, or animals or the
environment), while retaining stability when mixed with other
agents (e.g., a composition comprising an immunogen (e.g.,
bacteria, fungi, viruses, and spores). While a number of the above
described nanoemulsions meet these qualifications, the following
description provides a number of preferred non-toxic, non-irritant,
non-corrosive, anti-microbial nanoemulsions of the present
invention (hereinafter in this section referred to as "non-toxic
nanoemulsions").
[0139] In some embodiments the non-toxic nanoemulsions comprise
surfactant lipid preparations (SLPs) for use as broad-spectrum
antimicrobial agents that are effective against bacteria and their
spores, enveloped viruses, and fungi. In preferred embodiments,
these SLPs comprise a mixture of oils, detergents, solvents, and
cationic halogen-containing compounds in addition to several ions
that enhance their biocidal activities. These SLPs are
characterized as stable, non-irritant, and non-toxic compounds
compared to commercially available bactericidal and sporicidal
agents, which are highly irritant and/or toxic.
[0140] Ingredients for use in the non-toxic nanoemulsions include,
but are not limited to: detergents (e.g., TRITON X-100 (5-15%) or
other members of the TRITON family, TWEEN 60 (0.5-2%) or other
members of the TWEEN family, or TYLOXAPOL (1-10%)); solvents (e.g.,
tributyl phosphate (5-15%)); alcohols (e.g., ethanol (5-15%) or
glycerol (5-15%)); oils (e.g., soybean oil (40-70%)); cationic
halogen-containing compounds (e.g., cetylpyridinium chloride
(0.5-2%), cetylpyridinium bromide (0.5-2%)), or cetyldimethylethyl
ammonium bromide (0.5-2%)); quaternary ammonium compounds (e.g.,
benzalkonium chloride (0.5-2%), N-alkyldimethylbenzyl ammonium
chloride (0.5-2%)); ions (calcium chloride (1 mM-40 mM), ammonium
chloride (1 mM-20 mM), sodium chloride (5 mM-200 mM), sodium
phosphate (1 mM-20 mM)); nucleosides (e.g., inosine (50 .mu.M-20
mM)); and amino acids (e.g., L-alanine (50 .mu.M-20 mM)). Emulsions
are prepared, for example, by mixing in a high shear mixer for 3-10
minutes. The emulsions may or may not be heated before mixing at
82.degree. C. for 1 hour.
[0141] Quaternary ammonium compounds for use in the present
include, but are not limited to, N-alkyldimethyl benzyl ammonium
saccharinate; 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol;
1-Decanaminium, N-decyl-N, N-dimethyl-, chloride (or) Didecyl
dimethyl ammonium chloride; 2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ehyl
dimethyl benzyl ammonium chloride;
2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ammonium
chloride; alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazolinium
chloride; alkyl bis(2-hydroxyethyl) benzyl ammonium chloride; alkyl
demethyl benzyl ammonium chloride; alkyl dimethyl
3,4-dichlorobenzyl ammonium chloride (100% C12); alkyl dimethyl
3,4-dichlorobenzyl ammonium chloride (50% C14, 40% C12, 10% C16);
alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (55% C14, 23%
C12, 20% C16); alkyl dimethyl benzyl ammonium chloride; alkyl
dimethyl benzyl ammonium chloride (100% C14); alkyl dimethyl benzyl
ammonium chloride (100% C16); alkyl dimethyl benzyl ammonium
chloride (41% C14, 28% C12); alkyl dimethyl benzyl ammonium
chloride (47% C12, 18% C14); alkyl dimethyl benzyl ammonium
chloride (55% C16, 20% C14); alkyl dimethyl benzyl ammonium
chloride (58% C14, 28% C16); alkyl dimethyl benzyl ammonium
chloride (60% C14, 25% C12); alkyl dimethyl benzyl ammonium
chloride (61% C11, 23% C14); alkyl dimethyl benzyl ammonium
chloride (61% C12, 23% C14); alkyl dimethyl benzyl ammonium
chloride (65% C12, 25% C14); alkyl dimethyl benzyl ammonium
chloride (67% C12, 24% C14); alkyl dimethyl benzyl ammonium
chloride (67% C12, 25% C14); alkyl dimethyl benzyl ammonium
chloride (90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride
(93% C14, 4% C12); alkyl dimethyl benzyl ammonium chloride (95%
C16, 5% C18); alkyl dimethyl benzyl ammonium chloride (and) didecyl
dimethyl ammonium chloride; alkyl dimethyl benzyl ammonium chloride
(as in fatty acids); alkyl dimethyl benzyl ammonium chloride
(C12-C16); alkyl dimethyl benzyl ammonium chloride (C12-C18); alkyl
dimethyl benzyl and dialkyl dimethyl ammonium chloride; alkyl
dimethyl dimethybenzyl ammonium chloride; alkyl dimethyl ethyl
ammonium bromide (90% C14, 5% C16, 5% C12); alkyl dimethyl ethyl
ammonium bromide (mixed alkyl and alkenyl groups as in the fatty
acids of soybean oil); alkyl dimethyl ethylbenzyl ammonium
chloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14);
alkyl dimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14,
17% C16, 3% C18); alkyl trimethyl ammonium chloride (58% C18, 40%
C16, 1% C14, 1% C12); alkyl trimethyl ammonium chloride (90% C18,
10% C16); alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18);
Di-(C8-10)-alkyl dimethyl ammonium chlorides; dialkyl dimethyl
ammonium chloride; dialkyl dimethyl ammonium chloride; dialkyl
dimethyl ammonium chloride; dialkyl methyl benzyl ammonium
chloride; didecyl dimethyl ammonium chloride; diisodecyl dimethyl
ammonium chloride; dioctyl dimethyl ammonium chloride; dodecyl
bis(2-hydroxyethyl) octyl hydrogen ammonium chloride; dodecyl
dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyl dinethyl
benzyl ammonium chloride; heptadecyl hydroxyethylimidazolinium
chloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;
myristalkonium chloride (and) Quat RNIUM 14;
N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl
dimethyl benzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl
ammonium chloride; n-tetradecyl dimethyl benzyl ammonium chloride
monohydrate; octyl decyl dimethyl ammonium chloride; octyl dodecyl
dimethyl ammonium chloride; octyphenoxyethoxyethyl dimethyl benzyl
ammonium chloride; oxydiethylenebis(alkyl dimethyl ammonium
chloride); quaternary ammonium compounds, dicoco alkyldimethyl,
chloride; trimethoxysily propyl dimethyl octadecyl ammonium
chloride; trimethoxysilyl quats, trimethyl dodecylbenzyl ammonium
chloride; n-dodecyl dimethyl ethylbenzyl ammonium chloride;
n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyl
dimethyl benzyl ammonium chloride; n-tetradecyl dimethyl
ethyylbenzyl ammonium chloride; and n-octadecyl dimethyl benzyl
ammonium chloride.
[0142] 1. Aqueous Phase
[0143] In some embodiments, the emulsion comprises an aqueous
phase. In certain preferred embodiments, the emulsion comprises
about 5 to 50, preferably 10 to 40, more preferably 15 to 30, vol.
% aqueous phase, based on the total volume of the emulsion
(although other concentrations are also contemplated). In preferred
embodiments, the aqueous phase comprises water at a pH of about 4
to 10, preferably about 6 to 8. The water is preferably deionized
(hereinafter "DiH.sub.2O"). In some embodiments, the aqueous phase
comprises phosphate buffered saline (PBS). In some preferred
embodiments, the aqueous phase is sterile and pyrogen free.
[0144] 2. Oil Phase
[0145] In some embodiments, the emulsion comprises an oil phase. In
certain preferred embodiments, the oil phase (e.g., carrier oil) of
the emulsion of the present invention comprises 30-90, preferably
60-80, and more preferably 60-70, vol. % of oil, based on the total
volume of the emulsion (although higher and lower concentrations
also find use in emulsions described herein).
[0146] The oil in the nanoemulsion adjuvant of the invention can be
any cosmetically or pharmaceutically acceptable oil. The oil can be
volatile or non-volatile, and may be chosen from animal oil,
vegetable oil, natural oil, synthetic oil, hydrocarbon oils,
silicone oils, semi-synthetic derivatives thereof, and combinations
thereof.
[0147] Suitable oils include, but are not limited to, mineral oil,
squalene oil, flavor oils, silicon oil, essential oils, water
insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl
palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate,
Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl
salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols,
Ceraphyls.RTM., Decyl oleate, diisopropyl adipate, C.sub.12-15
alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl
neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate,
Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin,
Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil,
Chile oil, Coconut oil, corn oil, Cottonseed oil, Flaxseed oil,
Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil,
Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice
bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil,
Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil),
Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed
oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil,
Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, Avocado oil,
Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed
oil, almond seed oil, anise seed oil, celery seed oil, cumin seed
oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil,
cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon
grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli
leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil,
spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen
leaf oil, flower oil, chamomile oil, clary sage oil, clove oil,
geranium flower oil, hyssop flower oil, jasmine flower oil,
lavender flower oil, manuka flower oil, Marhoram flower oil, orange
flower oil, rose flower oil, ylang-ylang flower oil, Bark oil,
cassia Bark oil, cinnamon bark oil, sassafras Bark oil, Wood oil,
camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil),
rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil,
peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil,
lime peel oil, orange peel oil, tangerine peel oil, root oil,
valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl
alcohol, semi-synthetic derivatives thereof, and any combinations
thereof.
[0148] The oil may further comprise a silicone component, such as a
volatile silicone component, which can be the sole oil in the
silicone component or can be combined with other silicone and
non-silicone, volatile and non-volatile oils. Suitable silicone
components include, but are not limited to,
methylphenylpolysiloxane, simethicone, dimethicone,
phenyltrimethicone (or an organomodified version thereof),
alkylated derivatives of polymeric silicones, cetyl dimethicone,
lauryl trimethicone, hydroxylated derivatives of polymeric
silicones, such as dimethiconol, volatile silicone oils, cyclic and
linear silicones, cyclomethicone, derivatives of cyclomethicone,
hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane,
decamethylcyclopentasiloxane, volatile linear
dimethylpolysiloxanes, isohexadecane, isoeicosane, isotetracosane,
polyisobutene, isooctane, isododecane, semi-synthetic derivatives
thereof, and combinations thereof.
[0149] The volatile oil can be the organic solvent, or the volatile
oil can be present in addition to an organic solvent. Suitable
volatile oils include, but are not limited to, a terpene,
monoterpene, sesquiterpene, carminative, azulene, menthol, camphor,
thujone, thymol, nerol, linalool, limonene, geraniol, perillyl
alcohol, nerolidol, farnesol, ylangene, bisabolol, farnesene,
ascaridole, chenopodium oil, citronellal, citral, citronellol,
chamazulene, yarrow, guaiazulene, chamomile, semi-synthetic
derivatives, or combinations thereof.
[0150] In one aspect of the invention, the volatile oil in the
silicone component is different than the oil in the oil phase.
[0151] In some embodiments, the oil phase comprises 3-15, and
preferably 5-10 vol. % of an organic solvent, based on the total
volume of the emulsion. While the present invention is not limited
to any particular mechanism, it is contemplated that the organic
phosphate-based solvents employed in the emulsions serve to remove
or disrupt the lipids in the membranes of the pathogens. Thus, any
solvent that removes the sterols or phospholipids in the microbial
membranes finds use in the methods of the present invention.
Suitable organic solvents include, but are not limited to, organic
phosphate based solvents or alcohols. In some preferred
embodiments, non-toxic alcohols (e.g., ethanol) are used as a
solvent. The oil phase, and any additional compounds provided in
the oil phase, are preferably sterile and pyrogen free.
[0152] 3. Surfactants and Detergents
[0153] In some embodiments, the emulsions further comprises a
surfactant or detergent. In some preferred embodiments, the
emulsion comprises from about 3 to 15%, and preferably about 10% of
one or more surfactants or detergents (although other
concentrations are also contemplated). While the present invention
is not limited to any particular mechanism, it is contemplated that
surfactants, when present in the emulsions, help to stabilize the
emulsions. Both non-ionic (non-anionic) and ionic surfactants are
contemplated. Additionally, surfactants from the BRIJ family of
surfactants find use in the compositions of the present invention.
The surfactant can be provided in either the aqueous or the oil
phase. Surfactants suitable for use with the emulsions include a
variety of anionic and nonionic surfactants, as well as other
emulsifying compounds that are capable of promoting the formation
of oil-in-water emulsions. In general, emulsifying compounds are
relatively hydrophilic, and blends of emulsifying compounds can be
used to achieve the necessary qualities. In some formulations,
nonionic surfactants have advantages over ionic emulsifiers in that
they are substantially more compatible with a broad pH range and
often form more stable emulsions than do ionic (e.g., soap-type)
emulsifiers.
[0154] The surfactant in the nanoemulsion adjuvant of the invention
can be a pharmaceutically acceptable ionic surfactant, a
pharmaceutically acceptable nonionic surfactant, a pharmaceutically
acceptable cationic surfactant, a pharmaceutically acceptable
anionic surfactant, or a pharmaceutically acceptable zwitterionic
surfactant.
[0155] Exemplary useful surfactants are described in Applied
Surfactants: Principles and Applications. Tharwat F. Tadros,
Copyright 8 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-30629-3), which is specifically incorporated by
reference. Further, the surfactant can be a pharmaceutically
acceptable ionic polymeric surfactant, a pharmaceutically
acceptable nonionic polymeric surfactant, a pharmaceutically
acceptable cationic polymeric surfactant, a pharmaceutically
acceptable anionic polymeric surfactant, or a pharmaceutically
acceptable zwitterionic polymeric surfactant. Examples of polymeric
surfactants include, but are not limited to, a graft copolymer of a
poly(methyl methacrylate) backbone with multiple (at least one)
polyethylene oxide (PEO) side chain, polyhydroxystearic acid, an
alkoxylated alkyl phenol formaldehyde condensate, a polyalkylene
glycol modified polyester with fatty acid hydrophobes, a polyester,
semi-synthetic derivatives thereof, or combinations thereof.
[0156] Surface active agents or surfactants, are amphipathic
molecules that consist of a non-polar hydrophobic portion, usually
a straight or branched hydrocarbon or fluorocarbon chain containing
8-18 carbon atoms, attached to a polar or ionic hydrophilic
portion. The hydrophilic portion can be nonionic, ionic or
zwitterionic. The hydrocarbon chain interacts weakly with the water
molecules in an aqueous environment, whereas the polar or ionic
head group interacts strongly with water molecules via dipole or
ion-dipole interactions. Based on the nature of the hydrophilic
group, surfactants are classified into anionic, cationic,
zwitterionic, nonionic and polymeric surfactants.
[0157] Suitable surfactants include, but are not limited to,
ethoxylated nonylphenol comprising 9 to 10 units of ethyleneglycol,
ethoxylated undecanol comprising 8 units of ethyleneglycol,
polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20)
sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate,
polyoxyethylene (20) sorbitan monooleate, sorbitan monolaurate,
sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate,
ethoxylated hydrogenated ricin oils, sodium laurylsulfate, a
diblock copolymer of ethyleneoxyde and propyleneoxyde, Ethylene
Oxide-Propylene Oxide Block Copolymers, and tetra-functional block
copolymers based on ethylene oxide and propylene oxide, Glyceryl
monoesters, Glyceryl caprate, Glyceryl caprylate, Glyceryl cocate,
Glyceryl erucate, Glyceryl hydroxysterate, Glyceryl isostearate,
Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate, Glyceryl
myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate,
Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thiglycolate,
Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate,
Glyceryl disterate, Glyceryl sesuioleate, Glyceryl stearate
lactate, Polyoxyethylene cetyl/stearyl ether, Polyoxyethylene
cholesterol ether, Polyoxyethylene laurate or dilaurate,
Polyoxyethylene stearate or distearate, polyoxyethylene fatty
ethers, Polyoxyethylene lauryl ether, Polyoxyethylene stearyl
ether, polyoxyethylene myristyl ether, a steroid, Cholesterol,
Betasitosterol, Bisabolol, fatty acid esters of alcohols, isopropyl
myristate, Aliphati-isopropyl n-butyrate, Isopropyl n-hexanoate,
Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecyl
myristate, alkoxylated alcohols, alkoxylated acids, alkoxylated
amides, alkoxylated sugar derivatives, alkoxylated derivatives of
natural oils and waxes, polyoxyethylene polyoxypropylene block
copolymers, nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20
methylglucose sesquistearate, PEG40 lanolin, PEG-40 castor oil,
PEG-40 hydrogenated castor oil, polyoxyethylene fatty ethers,
glyceryl diesters, polyoxyethylene stearyl ether, polyoxyethylene
myristyl ether, and polyoxyethylene lauryl ether, glyceryl
dilaurate, glyceryl dimystate, glyceryl distearate, semi-synthetic
derivatives thereof, or mixtures thereof.
[0158] Additional suitable surfactants include, but are not limited
to, non-ionic lipids, such as glyceryl laurate, glyceryl myristate,
glyceryl dilaurate, glyceryl dimyristate, semi-synthetic
derivatives thereof, and mixtures thereof.
[0159] In additional embodiments, the surfactant is a
polyoxyethylene fatty ether having a polyoxyethylene head group
ranging from about 2 to about 100 groups, or an alkoxylated alcohol
having the structure R.sub.5--(OCH.sub.2 CH.sub.2).sub.y--OH,
wherein R.sub.5 is a branched or unbranched alkyl group having from
about 6 to about 22 carbon atoms and y is between about 4 and about
100, and preferably, between about 10 and about 100. Preferably,
the alkoxylated alcohol is the species wherein R.sub.5 is a lauryl
group and y has an average value of 23. In a different embodiment,
the surfactant is an alkoxylated alcohol which is an ethoxylated
derivative of lanolin alcohol. Preferably, the ethoxylated
derivative of lanolin alcohol is laneth-10, which is the
polyethylene glycol ether of lanolin alcohol with an average
ethoxylation value of 10.
[0160] Nonionic surfactants include, but are not limited to, an
ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol
ethoxylated, a fatty acid ethoxylated, a monoalkaolamide
ethoxylated, a sorbitan ester ethoxylated, a fatty amino
ethoxylated, an ethylene oxide-propylene oxide copolymer,
Bis(polyethylene glycol bis[imidazoyl carbonyl]), nonoxynol-9,
Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij.RTM. 35,
Brij.RTM. 56, Brij.RTM. 72, Brij.RTM. 76, Brij.RTM. 92V, Brij.RTM.
97, Brij.RTM. 58P, Cremophor.RTM. EL, Decaethylene glycol
monododecyl ether, N-Decanoyl-N-methylglucamine, n-Decyl
alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside,
n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside,
n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside,
Heptaethylene glycol monodecyl ether, Heptaethylene glycol
monododecyl ether, Heptaethylene glycol monotetradecyl ether,
n-Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl
ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol
monooctadecyl ether, Hexaethylene glycol monotetradecyl ether,
Igepal CA-630, Igepal CA-630,
Methyl-6-O-(N-heptylcarbamoyl)-alpha-D-glucopyranoside,
Nonaethylene glycol monododecyl ether,
N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N-methylglucamine,
Octaethylene glycol monodecyl ether, Octaethylene glycol
monododecyl ether, Octaethylene glycol monohexadecyl ether,
Octaethylene glycol monooctadecyl ether, Octaethylene glycol
monotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene
glycol monodecyl ether, Pentaethylene glycol monododecyl ether,
Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol
monohexyl ether, Pentaethylene glycol monooctadecyl ether,
Pentaethylene glycol monooctyl ether, Polyethylene glycol
diglycidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene 10
tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20
isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene
40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8
stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene
25 propylene glycol stearate, Saponin from Quillaja bark, Span.RTM.
20, Span.RTM. 40, Span.RTM. 60, Span.RTM. 65, Span.RTM. 80,
Span.RTM. 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30,
Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type
15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type
NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol,
Tergitol, Type TMN-10, Tergitol, Type TMN-6,
Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether,
Tetraethylene glycol monododecyl ether, Tetraethylene glycol
monotetradecyl ether, Triethylene glycol monodecyl ether,
Triethylene glycol monododecyl ether, Triethylene glycol
monohexadecyl ether, Triethylene glycol monooctyl ether,
Triethylene glycol monotetradecyl ether, Triton CF-21, Triton
CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15,
Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton
X-151, Triton X-200, Triton X-207, Triton.RTM. X-100, Triton.RTM.
X-114, Triton.RTM. X-165, Triton.RTM. X-305, Triton.RTM. X-405,
Triton.RTM. X-45, Triton.RTM. X-705-70, TWEEN.RTM. 20, TWEEN.RTM.
21, TWEEN.RTM. 40, TWEEN.RTM. 60, TWEEN.RTM. 61, TWEEN.RTM. 65,
TWEEN.RTM. 80, TWEEN.RTM. 81, TWEEN.RTM. 85, Tyloxapol, n-Undecyl
beta-D-glucopyranoside, semi-synthetic derivatives thereof, or
combinations thereof.
[0161] In addition, the nonionic surfactant can be a poloxamer.
Poloxamers are polymers made of a block of polyoxyethylene,
followed by a block of polyoxypropylene, followed by a block of
polyoxyethylene. The average number of units of polyoxyethylene and
polyoxypropylene varies based on the number associated with the
polymer. For example, the smallest polymer, Poloxamer 101, consists
of a block with an average of 2 units of polyoxyethylene, a block
with an average of 16 units of polyoxypropylene, followed by a
block with an average of 2 units of polyoxyethylene. Poloxamers
range from colorless liquids and pastes to white solids. In
cosmetics and personal care products, Poloxamers are used in the
formulation of skin cleansers, bath products, shampoos, hair
conditioners, mouthwashes, eye makeup remover and other skin and
hair products. Examples of Poloxamers include, but are not limited
to, Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122,
Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182,
Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188,
Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231,
Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238,
Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331,
Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338,
Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407,
Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.
[0162] Suitable cationic surfactants include, but are not limited
to, a quarternary ammonium compound, an alkyl trimethyl ammonium
chloride compound, a dialkyl dimethyl ammonium chloride compound, a
cationic halogen-containing compound, such as cetylpyridinium
chloride, Benzalkonium chloride, Benzalkonium chloride,
Benzyldimethylhexadecylammonium chloride,
Benzyldimethyltetradecylammonium chloride,
Benzyldodecyldimethylammonium bromide, Benzyltrimethylammonium
tetrachloroiodate, Dimethyldioctadecylammonium bromide,
Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium
bromide, Dodecyltrimethylammonium bromide,
Ethylhexadecyldimethylammonium bromide, Girard's reagent T,
Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium
bromide, N,N',N'-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane,
Thonzonium bromide, Trimethyl(tetradecyl)ammonium bromide,
1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, 1-Decanaminium,
N-decyl-N, N-dimethyl-, chloride, Didecyl dimethyl ammonium
chloride, 2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl
ammonium chloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl
dimethyl benzyl ammonium chloride, Alkyl 1 or 3
benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride, Alkyl
bis(2-hydroxyethyl) benzyl ammonium chloride, Alkyl demethyl benzyl
ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (100% C12), Alkyl dimethyl 3,4-dichlorobenzyl ammonium
chloride (50% C14, 40% C12, 10% C16), Alkyl dimethyl
3,4-dichlorobenzyl ammonium chloride (55% C14, 23% C12, 20% C16),
Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyl
ammonium chloride (100% C14), Alkyl dimethyl benzyl ammonium
chloride (100% C16), Alkyl dimethyl benzyl ammonium chloride (41%
C14, 28% C12), Alkyl dimethyl benzyl ammonium chloride (47% C12,
18% C14), Alkyl dimethyl benzyl ammonium chloride (55% C16, 20%
C14), Alkyl dimethyl benzyl ammonium chloride (58% C14, 28% C16),
Alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12), Alkyl
dimethyl benzyl ammonium chloride (61% C11, 23% C14), Alkyl
dimethyl benzyl ammonium chloride (61% C12, 23% C14), Alkyl
dimethyl benzyl ammonium chloride (65% C12, 25% C14), Alkyl
dimethyl benzyl ammonium chloride (67% C12, 24% C14), Alkyl
dimethyl benzyl ammonium chloride (67% C12, 25% C14), Alkyl
dimethyl benzyl ammonium chloride (90% C14, 5% C12), Alkyl dimethyl
benzyl ammonium chloride (93% C14, 4% C12), Alkyl dimethyl benzyl
ammonium chloride (95% C16, 5% C18), Alkyl dimethyl benzyl ammonium
chloride, Alkyl didecyl dimethyl ammonium chloride, Alkyl dimethyl
benzyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride
(C12-16), Alkyl dimethyl benzyl ammonium chloride (C12-18), Alkyl
dimethyl benzyl ammonium chloride, dialkyl dimethyl benzyl ammonium
chloride, Alkyl dimethyl dimethybenzyl ammonium chloride, Alkyl
dimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12), Alkyl
dimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as
in the fatty acids of soybean oil), Alkyl dimethyl ethylbenzyl
ammonium chloride, Alkyl dimethyl ethylbenzyl ammonium chloride
(60% C14), Alkyl dimethyl isopropylbenzyl ammonium chloride (50%
C12, 30% C14, 17% C16, 3% C18), Alkyl trimethyl ammonium chloride
(58% C18, 40% C16, 1% C14, 1% C12), Alkyl trimethyl ammonium
chloride (90% C18, 10% C16), Alkyldimethyl(ethylbenzyl) ammonium
chloride (C12-18), Di-(C8-10)-alkyl dimethyl ammonium chlorides,
Dialkyl dimethyl ammonium chloride, Dialkyl methyl benzyl ammonium
chloride, Didecyl dimethyl ammonium chloride, Diisodecyl dimethyl
ammonium chloride, Dioctyl dimethyl ammonium chloride, Dodecyl
bis(2-hydroxyethyl) octyl hydrogen ammonium chloride, Dodecyl
dimethyl benzyl ammonium chloride, Dodecylcarbamoyl methyl dinethyl
benzyl ammonium chloride, Heptadecyl hydroxyethylimidazolinium
chloride, Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine,
Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium
chloride (and) Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammonium
chloride polymer, n-Tetradecyl dimethyl benzyl ammonium chloride
monohydrate, Octyl decyl dimethyl ammonium chloride, Octyl dodecyl
dimethyl ammonium chloride, Octyphenoxyethoxyethyl dimethyl benzyl
ammonium chloride, Oxydiethylenebis(alkyl dimethyl ammonium
chloride), Quaternary ammonium compounds, dicoco alkyldimethyl,
chloride, Trimethoxysily propyl dimethyl octadecyl ammonium
chloride, Trimethoxysilyl quats, Trimethyl dodecylbenzyl ammonium
chloride, semi-synthetic derivatives thereof, and combinations
thereof.
[0163] Exemplary cationic halogen-containing compounds include, but
are not limited to, cetylpyridinium halides, cetyltrimethylammonium
halides, cetyldimethylethylammonium halides,
cetyldimethylbenzylammonium halides, cetyltributylphosphonium
halides, dodecyltrimethylammonium halides, or
tetradecyltrimethylammonium halides. In some particular
embodiments, suitable cationic halogen containing compounds
comprise, but are not limited to, cetylpyridinium chloride (CPC),
cetyltrimethylammonium chloride, cetylbenzyldimethylammonium
chloride, cetylpyridinium bromide (CPB), cetyltrimethylammonium
bromide (CTAB), cetyidimethylethylammonium bromide,
cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide,
and tetrad ecyltrimethylammonium bromide. In particularly preferred
embodiments, the cationic halogen containing compound is CPC,
although the compositions of the present invention are not limited
to formulation with an particular cationic containing compound.
[0164] Suitable anionic surfactants include, but are not limited
to, a carboxylate, a sulphate, a sulphonate, a phosphate,
chenodeoxycholic acid, chenodeoxycholic acid sodium salt, cholic
acid, ox or sheep bile, Dehydrocholic acid, Deoxycholic acid,
Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin,
Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusate sodium
salt, Glycochenodeoxycholic acid sodium salt, Glycocholic acid
hydrate, synthetic, Glycocholic acid sodium salt hydrate,
synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid
sodium salt, Glycodeoxycholic acid sodium salt, Glycolithocholic
acid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester,
N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine solution,
N-Lauroylsarcosine solution, Lithium dodecyl sulfate, Lithium
dodecyl sulfate, Lithium dodecyl sulfate, Lugol solution, Niaproof
4, Type 4, 1-Octanesulfonic acid sodium salt, Sodium
1-butanesulfonate, Sodium 1-decanesulfonate, Sodium
1-decanesulfonate, Sodium 1-dodecanesulfonate, Sodium
1-heptanesulfonate anhydrous, Sodium 1-heptanesulfonate anhydrous,
Sodium 1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate,
Sodium 2-bromoethanesulfonate, Sodium cholate hydrate, Sodium
choleate, Sodium deoxycholate, Sodium deoxycholate monohydrate,
Sodium dodecyl sulfate, Sodium hexanesulfonate anhydrous, Sodium
octyl sulfate, Sodium pentanesulfonate anhydrous, Sodium
taurocholate, Taurochenodeoxycholic acid sodium salt,
Taurodeoxycholic acid sodium salt monohydrate, Taurohyodeoxycholic
acid sodium salt hydrate, Taurolithocholic acid 3-sulfate disodium
salt, Tauroursodeoxycholic acid sodium salt, Trizma.RTM. dodecyl
sulfate, TWEEN.RTM. 80, Ursodeoxycholic acid, semi-synthetic
derivatives thereof, and combinations thereof.
[0165] Suitable zwitterionic surfactants include, but are not
limited to, an N-alkyl betaine, lauryl amindo propyl dimethyl
betaine, an alkyl dimethyl glycinate, an N-alkyl amino propionate,
CHAPS, minimum 98% (TLC), CHAPS, SigmaUltra, minimum 98% (TLC),
CHAPS, for electrophoresis, minimum 98% (TLC), CHAPSO, minimum 98%,
CHAPSO, SigmaUltra, CHAPSO, for electrophoresis,
3-(Decyldimethylammonio)propanesulfonate inner salt,
3-Dodecyldimethylammonio)propanesulfonate inner salt, SigmaUltra,
3-(Dodecyldimethylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylmyristylammonio)propanesulfonate,
3-(N,N-Dimethyloctadecylammonio)propanesulfonate,
3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,
3-(N,N-Dimethylpalmitylammonio)propanesulfonate, semi-synthetic
derivatives thereof, and combinations thereof.
[0166] The present invention is not limited to the surfactants
disclosed herein. Additional surfactants and detergents useful in
the compositions of the present invention may be ascertained from
reference works (e.g., including, but not limited to, McCutheon's
Volume 1: Emulsions and Detergents--North American Edition, 2000)
and commercial sources.
[0167] 4. Cationic Halogens Containing Compounds
[0168] In some embodiments, the emulsions further comprise a
cationic halogen containing compound. In some preferred
embodiments, the emulsion comprises from about 0.5 to 1.0 wt. % or
more of a cationic halogen containing compound, based on the total
weight of the emulsion (although other concentrations are also
contemplated). In preferred embodiments, the cationic
halogen-containing compound is preferably premixed with the oil
phase; however, it should be understood that the cationic
halogen-containing compound may be provided in combination with the
emulsion composition in a distinct formulation. Suitable halogen
containing compounds may be selected from compounds comprising
chloride, fluoride, bromide and iodide ions. In preferred
embodiments, suitable cationic halogen containing compounds
include, but are not limited to, cetylpyridinium halides,
cetyltrimethylammonium halides, cetyldimethylethylammonium halides,
cetyldimethylbenzylammonium halides, cetyltributylphosphonium
halides, dodecyltrimethylammonium halides, or
tetradecyltrimethylammonium halides. In some particular
embodiments, suitable cationic halogen containing compounds
comprise, but are not limited to, cetylpyridinium chloride (CPC),
cetyltrimethylammonium chloride, cetylbenzyldimethylammonium
chloride, cetylpyridinium bromide (CPB), and cetyltrimethylammonium
bromide (CTAB), cetyidimethylethylammonium bromide,
cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide,
and tetrad ecyltrimethylammonium bromide. In particularly preferred
embodiments, the cationic halogen-containing compound is CPC,
although the compositions of the present invention are not limited
to formulation with any particular cationic containing
compound.
[0169] 5. Germination Enhancers
[0170] In other embodiments of the present invention, the
nanoemulsions further comprise a germination enhancer. In some
preferred embodiments, the emulsions comprise from about 1 mM to 15
mM, and more preferably from about 5 mM to 10 mM of one or more
germination enhancing compounds (although other concentrations are
also contemplated). In preferred embodiments, the germination
enhancing compound is provided in the aqueous phase prior to
formation of the emulsion. The present invention contemplates that
when germination enhancers are added to the nanoemulsion
compositions, the sporicidal properties of the nanoemulsions are
enhanced. The present invention further contemplates that such
germination enhancers initiate sporicidal activity near neutral pH
(between pH 6-8, and preferably 7). Such neutral pH emulsions can
be obtained, for example, by diluting with phosphate buffer saline
(PBS) or by preparations of neutral emulsions. The sporicidal
activity of the nanoemulsion preferentially occurs when the spores
initiate germination.
[0171] In specific embodiments, it has been demonstrated that the
emulsions utilized in the vaccines of the present invention have
sporicidal activity. While the present invention is not limited to
any particular mechanism and an understanding of the mechanism is
not required to practice the present invention, it is believed that
the fusigenic component of the emulsions acts to initiate
germination and before reversion to the vegetative form is complete
the lysogenic component of the emulsion acts to lyse the newly
germinating spore. These components of the emulsion thus act in
concert to leave the spore susceptible to disruption by the
emulsions. The addition of germination enhancer further facilitates
the anti-sporicidal activity of the emulsions, for example, by
speeding up the rate at which the sporicidal activity occurs.
[0172] Germination of bacterial endospores and fungal spores is
associated with increased metabolism and decreased resistance to
heat and chemical reactants. For germination to occur, the spore
must sense that the environment is adequate to support vegetation
and reproduction. The amino acid L-alanine stimulates bacterial
spore germination (See e.g., Hills, J. Gen. Micro. 4:38 (1950); and
Halvorson and Church, Bacteriol Rev. 21:112 (1957)). L-alanine and
L-proline have also been reported to initiate fungal spore
germination (Yanagita, Arch Mikrobiol 26:329 (1957)). Simple
.alpha.-amino acids, such as glycine and L-alanine, occupy a
central position in metabolism. Transamination or deamination of
.alpha.-amino acids yields the glycogenic or ketogenic
carbohydrates and the nitrogen needed for metabolism and growth.
For example, transamination or deamination of L-alanine yields
pyruvate, which is the end product of glycolytic metabolism
(EmBECTON DICKENSONen-Meyerhof-Parnas Pathway). Oxidation of
pyruvate by pyruvate dehydrogenase complex yields acetyl-CoA, NADH,
H.sup.+, and CO.sub.2. Acetyl-CoA is the initiator substrate for
the tricarboxylic acid cycle (Kreb's Cycle), which in turns feeds
the mitochondrial electron transport chain. Acetyl-CoA is also the
ultimate carbon source for fatty acid synthesis as well as for
sterol synthesis. Simple .alpha.-amino acids can provide the
nitrogen, CO.sub.2, glycogenic and/or ketogenic equivalents
required for germination and the metabolic activity that
follows.
[0173] In certain embodiments, suitable germination enhancing
agents of the invention include, but are not limited to,
.alpha.-amino acids comprising glycine and the L-enantiomers of
alanine, valine, leucine, isoleucine, serine, threonine, lysine,
phenylalanine, tyrosine, and the alkyl esters thereof. Additional
information on the effects of amino acids on germination may be
found in U.S. Pat. No. 5,510,104; herein incorporated by reference
in its entirety. In some embodiments, a mixture of glucose,
fructose, asparagine, sodium chloride (NaCl), ammonium chloride
(NH.sub.4Cl), calcium chloride (CaCl.sub.2) and potassium chloride
(KCl) also may be used. In particularly preferred embodiments of
the present invention, the formulation comprises the germination
enhancers L-alanine, CaCl.sub.2, Inosine and NH.sub.4Cl. In some
embodiments, the compositions further comprise one or more common
forms of growth media (e.g., trypticase soy broth, and the like)
that additionally may or may not itself comprise germination
enhancers and buffers.
[0174] The above compounds are merely exemplary germination
enhancers and it is understood that other known germination
enhancers will find use in the nanoemulsions utilized in some
embodiments of the present invention. A candidate germination
enhancer should meet two criteria for inclusion in the compositions
of the present invention: it should be capable of being associated
with the emulsions disclosed herein and it should increase the rate
of germination of a target spore when incorporated in the emulsions
disclosed herein. One skilled in the art can determine whether a
particular agent has the desired function of acting as an
germination enhancer by applying such an agent in combination with
the nanoemulsions disclosed herein to a target and comparing the
inactivation of the target when contacted by the admixture with
inactivation of like targets by the composition of the present
invention without the agent. Any agent that increases germination,
and thereby decreases or inhibits the growth of the organisms, is
considered a suitable enhancer for use in the nanoemulsion
compositions disclosed herein.
[0175] In still other embodiments, addition of a germination
enhancer (or growth medium) to a neutral emulsion composition
produces a composition that is useful in inactivating bacterial
spores in addition to enveloped viruses, Gram negative bacteria,
and Gram positive bacteria for use in the vaccine compositions of
the present invention.
[0176] 6. Interaction Enhancers
[0177] In still other embodiments, nanoemulsions comprise one or
more compounds capable of increasing the interaction of the
compositions (i.e., "interaction enhancer" (e.g., with target
pathogens (e.g., the cell wall of Gram negative bacteria such as
Vibrio, Salmonella, Shigella and Pseudomonas)). In preferred
embodiments, the interaction enhancer is preferably premixed with
the oil phase; however, in other embodiments the interaction
enhancer is provided in combination with the compositions after
emulsification. In certain preferred embodiments, the interaction
enhancer is a chelating agent (e.g., ethylenediaminetetraacetic
acid (EDTA) or ethylenebis(oxyethylenenitrilo)tetraacetic acid
(EGTA) in a buffer (e.g., tris buffer)). It is understood that
chelating agents are merely exemplary interaction enhancing
compounds. Indeed, other agents that increase the interaction of
the nanoemulsions used in some embodiments of the present invention
(e.g., with microbial agents, pathogens, vaccines, etc.) are
contemplated. In particularly preferred embodiments, the
interaction enhancer is at a concentration of about 50 to about 250
.mu.M. One skilled in the art will be able to determine whether a
particular agent has the desired function of acting as an
interaction enhancer by applying such an agent in combination with
the compositions of the present invention to a target and comparing
the inactivation of the target when contacted by the admixture with
inactivation of like targets by the composition of the present
invention without the agent. Any agent that increases the
interaction of an emulsion with bacteria and thereby decreases or
inhibits the growth of the bacteria, in comparison to that
parameter in its absence, is considered an interaction
enhancer.
[0178] In some embodiments, the addition of an interaction enhancer
to nanoemulsion produces a composition that is useful in
inactivating enveloped viruses, some Gram positive bacteria and
some Gram negative bacteria for use in a vaccine composition.
[0179] 7. Quaternary Ammonium Compounds
[0180] In some embodiments, nanoemulsions of the present invention
include a quaternary ammonium containing compound. Exemplary
quaternary ammonium compounds include, but are not limited to,
Alkyl dimethyl benzyl ammonium chloride, didecyl dimethyl ammonium
chloride, Alkyl dimethyl benzyl and dialkyl dimethyl ammonium
chloride, N,N-Dimethyl-2-hydroxypropylammonium chloride polymer,
Didecyl dimethyl ammonium chloride, n-Alkyl dimethyl benzyl
ammonium chloride, n-Alkyl dimethyl ethylbenzyl ammonium chloride,
Dialkyl dimethyl ammonium chloride, n-Alkyl dimethyl benzyl
ammonium chloride, n-Tetradecyl dimethyl benzyl ammonium chloride
monohydrate, n-Alkyl dimethyl benzyl ammonium chloride, Dialkyl
dimethyl ammonium chloride,
Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium
chloride (and) Quat RNIUM 14, Alkyl bis(2-hydroxyethyl) benzyl
ammonium chloride, Alkyl demethyl benzyl ammonium chloride, Alkyl
dimethyl 3,4-dichlorobenzyl ammonium chloride, Alkyl dimethyl
benzyl ammonium chloride, Alkyl dimethyl benzyl dimethylbenzyl
ammonium, Alkyl dimethyl dimethybenzyl ammonium chloride, Alkyl
dimethyl ethyl ammonium bromide, Alkyl dimethyl ethyl ammonium
bromide, Alkyl dimethyl ethylbenzyl ammonium chloride, Alkyl
dimethyl isopropylbenzyl ammonium chloride, Alkyl trimethyl
ammonium chloride, Alkyl 1 or 3
benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride, Dialkyl methyl
benzyl ammonium chloride, Dialkyl dimethyl ammonium chloride,
Didecyl dimethyl ammonium chloride,
2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammonium
chloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl
ammonium chloride, Dioctyl dimethyl ammonium chloride, Dodecyl
bis(2-hydroxyethyl) octyl hydrogen ammonium chloride, Dodecyl
dimethyl benzyl ammonium chloride, Dodecylcarbamoyl methyl dinethyl
benzyl ammonium chloride, Heptadecyl hydroxyethylimidazolinium
chloride, Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Octyl
decyl dimethyl ammonium chloride, Octyl dodecyl dimethyl ammonium
chloride, Octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride,
Oxydiethylenebis(alkyl dimethyl ammonium chloride), Quaternary
ammonium compounds, dicoco alkyldimethyl, chloride, Trimethoxysilyl
quats, and Trimethyl dodecylbenzyl ammonium chloride.
[0181] 8. Other Components
[0182] In some embodiments, a nanoemulsion adjuvant composition
comprises one or more additional components that provide a desired
property or functionality to the nanoemulsions. These components
may be incorporated into the aqueous phase or the oil phase of the
nanoemulsions and/or may be added prior to or following
emulsification. For example, in some embodiments, the nanoemulsions
further comprise phenols (e.g., triclosan, phenyl phenol),
acidifying agents (e.g., citric acid (e.g., 1.5-6%), acetic acid,
lemon juice), alkylating agents (e.g., sodium hydroxide (e.g.,
0.3%)), buffers (e.g., citrate buffer, acetate buffer, and other
buffers useful to maintain a specific pH), and halogens (e.g.,
polyvinylpyrrolidone, sodium hypochlorite, hydrogen peroxide).
[0183] Exemplary techniques for making a nanoemulsion are described
below. Additionally, a number of specific, although exemplary,
formulation recipes are also set forth herein.
[0184] In some embodiments, a nanoemulsion adjuvant is administered
to a subject before, concurrent with or after administration of a
composition comprising an immunogen (e.g., a pathogen and/or
pathogen component (e.g., purified, isolated and/or recombinant
pathogen peptide and/or protein)). The invention is not limited to
the use of any one specific type of composition comprising an
immunogen. Indeed, a variety of compositions comprising an
immunogen (e.g., utilized for generating an immune response (e.g.,
for use as a vaccine)) may be utilized with a nanoemulsion adjuvant
of the invention. In some embodiments, the composition comprising
an immunogen comprises pathogens (e.g., killed pathogens), pathogen
components or isolated, purified and/or recombinant parts
thereof.
[0185] In some embodiments, a nanoemulsion adjuvant is administered
to a subject before, concurrent with or after administration of a
vaccine containing peptides (e.g., one generally well known in the
art, as exemplified by U.S. Pat. Nos. 4,601,903; 4,599,231;
4,599,230; and 4,596,792; each of which is hereby incorporated by
reference).
Formulation Techniques
[0186] Nanoemulsions of the present invention can be formed using
classic emulsion forming techniques. In brief, the oil phase is
mixed with the aqueous phase under relatively high shear forces
(e.g., using high hydraulic and mechanical forces) to obtain an
oil-in-water nanoemulsion. The emulsion is formed by blending the
oil phase with an aqueous phase on a volume-to-volume basis ranging
from about 1:9 to 5:1, preferably about 5:1 to 3:1, most preferably
4:1, oil phase to aqueous phase. The oil and aqueous phases can be
blended using any apparatus capable of producing shear forces
sufficient to form an emulsion such as French Presses or high shear
mixers (e.g., FDA approved high shear mixers are available, for
example, from Admix, Inc., Manchester, N.H.). Methods of producing
such emulsions are described in U.S. Pat. Nos. 5,103,497 and
4,895,452, and U.S. Patent Application Nos. 20070036831,
20060251684, and 20050208083, herein incorporated by reference in
their entireties.
[0187] In preferred embodiments, compositions used in the methods
of the present invention comprise droplets of an oily discontinuous
phase dispersed in an aqueous continuous phase, such as water. In
preferred embodiments, nanoemulsions of the present invention are
stable, and do not decompose even after long storage periods (e.g.,
greater than one or more years). Furthermore, in some embodiments,
nanoemulsions are stable (e.g., in some embodiments for greater
than 3 months, in some embodiments for greater than 6 months, in
some embodiments for greater than 12 months, in some embodiments
for greater than 18 months) after combination with an immunogen. In
preferred embodiments, nanoemulsions of the present invention are
non-toxic and safe when administered (e.g., via spraying or
contacting mucosal surfaces, swallowed, inhaled, etc.) to a
subject.
[0188] In some embodiments, a portion of the emulsion may be in the
form of lipid structures including, but not limited to,
unilamellar, multilamellar, and paucliamellar lipid vesicles,
micelles, and lamellar phases.
[0189] As described above, the present invention is not limited by
the type of subject administered a composition of the present
invention. Indeed, a wide variety of subjects are contemplated to
be benefited from administration of a composition of the present
invention. In preferred embodiments, the subject is a human. In
some embodiments, human subjects are of any age (e.g., adults,
children, infants, etc.) that have been or are likely to become
exposed to a microorganism. In some embodiments, the human subjects
are subjects that are more likely to receive a direct exposure to
pathogenic microorganisms or that are more likely to display signs
and symptoms of disease after exposure to a pathogen (e.g.,
subjects with CF or asthma, subjects in the armed forces,
government employees, frequent travelers, persons attending or
working in a school or daycare, health care workers, an elderly
person, an immunocompromised person, and emergency service
employees (e.g., police, fire, EMT employees)). In some
embodiments, any one or all members of the general public can be
administered a composition of the present invention (e.g., to
prevent the occurrence or spread of disease). For example, in some
embodiments, compositions and methods of the present invention are
utilized to treat a group of people (e.g., a population of a
region, city, state and/or country) for their own health (e.g., to
prevent or treat disease) and/or to prevent or reduce the risk of
disease spread from animals (e.g., birds, cattle, sheep, pigs,
etc.) to humans. In some embodiments, the subjects are non-human
mammals (e.g., pigs, cattle, goats, horses, sheep, or other
livestock; or mice, rats, rabbits or other animal). In some
embodiments, compositions and methods of the present invention are
utilized in research settings (e.g., with research animals).
[0190] A composition comprising a nanoemulsion of the present
invention can be administered (e.g., to a subject (e.g., via
pulmonary and/or mucosal route)) as a therapeutic or as a
prophylactic to prevent microbial infection.
Therapeutics and Prophylactics
[0191] Furthermore, in preferred embodiments, a composition of the
present invention induces (e.g., when administered to a subject)
both systemic and mucosal immunity. Thus, in some preferred
embodiments, administration of a composition comprising a
nanoemulsion and Streptococcus (e.g., Streptococcus pneumoniae)
antigen to a subject results in protection against an exposure
(e.g., a mucosal exposure) to Streptococcus. Although an
understanding of the mechanism is not necessary to practice the
present invention and the present invention is not limited to any
particular mechanism of action, mucosal administration (e.g.,
vaccination) provides protection against Streptococcus (e.g.,
Streptococcus pneumoniae) infection (e.g., that initiates at a
mucosal surface). Although it has heretofore proven difficult to
stimulate secretory IgA responses and protection against pathogens
that invade at mucosal surfaces (See, e.g., Mestecky et al, Mucosal
Immunology. 3ed edn. (Academic Press, San Diego, 2005)), in some
embodiments, the present invention provides compositions and
methods for stimulating mucosal immunity (e.g., a protective IgA
response) from a pathogen (e.g., pathogenic species of
Streptococcus (e.g., Streptococcus pneumoniae)) in a subject.
[0192] In some embodiments, the present invention provides a
composition (e.g., a composition comprising a nanoemulsion and
Streptococcus (e.g., Streptococcus pneumoniae) antigen) to serve as
a mucosal vaccine. In some embodiments, this material is produced
with NE and killed whole cell bacteria of the genus Streptococcus
(e.g., Streptococcus pneumoniae (e.g., killed using nanoemulsion,
alcohol (e.g., ethanol), or other methods), isolated, purified
and/or recombinant protein and/or saccharide component of
Streptococcus (e.g., protein/peptide (e.g., Streptococcus-derived
protein, live-virus-vector-derived protein, recombinant protein,
recombinant denatured protein/antigens, small peptide segments
protein/antigen). The ability to produce this formulation rapidly
and administer it via mucosal (e.g., nasal) instillation provides a
vaccine that can be used in large-scale administrations (e.g., to a
population of a town, village, city, state or country). The present
invention is not limited to any particular formulation (e.g.,
comprising a nanoemulsion and one or more recombinant Streptococcus
proteins). For example, in some embodiments, the invention provides
a nanoemulsion described herein combined with one or more
recombinant Streptococcus proteins (e.g., PsaA, PiuA, PavA).
[0193] In some preferred embodiments, the present invention
provides a composition for generating an immune response comprising
a NE and an immunogen (e.g., a purified, isolated or synthetic
Streptococcus protein or derivative, variant, or analogue thereof;
or, one or more serotypes of Streptococcus (e.g., Streptococcus
pneumoniae (e.g., killed and or inactivated whole cell bacteria).
When administered to a subject, a composition of the present
invention stimulates an immune response against the immunogen
within the subject. Although an understanding of the mechanism is
not necessary to practice the present invention and the present
invention is not limited to any particular mechanism of action, in
some embodiments, generation of an immune response (e.g., resulting
from administration of a composition comprising a nanoemulsion and
an immunogen) provides total or partial immunity to the subject
(e.g., from signs, symptoms or conditions of a disease (e.g., strep
throat, meningitis, bacterial pneumoniae, endocarditis, erysipelas
and/or necrotizing fasciitis)). Without being bound to any specific
theory, protection and/or immunity from disease (e.g., the ability
of a subject's immune system to prevent or attenuate (e.g.,
suppress) a sign, symptom or condition of disease) after exposure
to an immunogenic composition of the present invention is due to
adaptive (e.g., acquired) immune responses (e.g., immune responses
mediated by B and T cells following exposure to a NE comprising an
immunogen of the present invention (e.g., immune responses that
exhibit increased specificity and reactivity towards Streptococcus
(e.g., Streptococcus pneumoniae)). Thus, in some embodiments, the
compositions and methods of the present invention are used
prophylactically or therapeutically to prevent or attenuate a sign,
symptom or condition associated with Streptococcus (e.g.,
Streptococcus pneumoniae)).
[0194] In some embodiments, a NE comprising an immunogen (e.g., a
Streptococcus (e.g., Streptococcus pneumoniae) antigen) is
administered alone. In some embodiments, a composition comprising a
NE and an immunogen (e.g., a Streptococcus (e.g., Streptococcus
pneumoniae) antigen) comprises one or more other agents (e.g., a
pharmaceutically acceptable carrier, adjuvant, excipient, and the
like). In some embodiments, a composition for stimulating an immune
response of the present invention is administered in a manner to
induce a humoral immune response. In some embodiments, a
composition for stimulating an immune response of the present
invention is administered in a manner to induce a cellular (e.g.,
cytotoxic T lymphocyte) immune response, rather than a humoral
response. In some embodiments, a composition comprising a NE and an
immunogen of the present invention induces both a cellular and
humoral immune response.
[0195] The present invention is not limited by the isotype or
strain of Streptococcus (e.g., Streptococcus pneumoniae) used in a
composition comprising a NE and immunogen. Indeed, each
Streptococcus (e.g., Streptococcus pneumoniae) family member alone,
or in combination with another family member, may be used to
generate a composition comprising a NE and an immunogen (e.g., used
to generate an immune response) of the present invention. Exemplary
species of Streptococcus and isotypes of Streptococcus pneumoniae
are described herein.
[0196] Thus, in some embodiments, the Streptococcus (e.g.,
Streptococcus pneumoniae) strain utilized is a modified (e.g.,
genetically modified (e.g., naturally modified via natural
selection or modified using recombinant genetic techniques)) strain
that displays greater pathogenic capacity (e.g., causes more sever
Streptococcus- (e.g., Streptococcus pneumoniae)-induced disease
(e.g., comprising enhanced and/or more severe strep throat,
meningitis, etc.)). In some embodiments, any one or more members of
the Streptococcus genus is utilized in an immunoreactive
composition of the invention including but not limited to S.
pneumoniae, S. mutans, S. mitis, S. sanguinis, S. salivarius, S.
viridans, S. salivarius ssp. thermophilus, S. constellatus, S.
pyogenes, S. agalactiae, S. zooepidemicus, Streptococcus bovis and
Streptococcus equines, Streptococcus canis, as well as former Group
D Streptococci including S. faecalis, S. faecium, S. durans, and S.
avium.
[0197] The present invention is not limited by the Streptococcus
(e.g., Streptococcus pneumoniae) isotype and/or strain used.
Indeed, a variety of Streptococcus (e.g., Streptococcus pneumoniae)
strains are contemplated to be useful in the present invention
including, but not limited to, classical strains, attenuated
strains, non-replicating strains, modified strains (e.g.,
genetically or mechanically modified strains (e.g., to become more
or less virulent)), or other serially diluted strains of
Streptococcus (e.g., Streptococcus pneumoniae). A composition
comprising a NE and immunogen may comprise one or more strains of
Streptococcus (e.g., Streptococcus pneumoniae) and/or other type of
Streptococcus (e.g., Streptococcus pneumoniae). Additionally, a
composition comprising a NE and immunogen may comprise one or more
strains of Streptococcus (e.g., Streptococcus pneumoniae), and, in
addition, one or more strains of a non-Streptococcus (e.g.,
Streptococcus pneumoniae) immunogen.
[0198] In some embodiments, the immunogen may comprise one or more
antigens derived from a pathogen (e.g., Streptococcus (e.g.,
Streptococcus pneumoniae)). For example, in some embodiments, the
immunogen is a purified, recombinant, synthetic, or otherwise
isolated protein (e.g., added to the NE to generate an immunogenic
composition). Similarly, the immunogenic protein may be a
derivative, analogue or otherwise modified (e.g., PEGylated) form
of a protein from a pathogen.
[0199] The present invention is not limited by the particular
formulation of a composition comprising a NE and immunogen of the
present invention. Indeed, a composition comprising a NE and
immunogen of the present invention may comprise one or more
different agents in addition to the NE and immunogen. These agents
or cofactors include, but are not limited to, adjuvants,
surfactants, additives, buffers, solubilizers, chelators, oils,
salts, therapeutic agents, drugs, bioactive agents, antibacterials,
and antimicrobial agents (e.g., antibiotics, antivirals, etc.). In
some embodiments, a composition comprising a NE and immunogen of
the present invention comprises an agent and/or co-factor that
enhance the ability of the immunogen to induce an immune response
(e.g., an adjuvant). In some preferred embodiments, the presence of
one or more co-factors or agents reduces the amount of immunogen
required for induction of an immune response (e.g., a protective
immune response (e.g., protective immunization)). In some
embodiments, the presence of one or more co-factors or agents can
be used to skew the immune response towards a cellular (e.g., T
cell mediated) or humoral (e.g., antibody mediated) immune
response. The present invention is not limited by the type of
co-factor or agent used in a therapeutic agent of the present
invention.
[0200] Adjuvants are described in general in Vaccine Design--the
Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum
Press, New York, 1995. The present invention is not limited by the
type of adjuvant utilized (e.g., for use in a composition (e.g.,
pharmaceutical composition) comprising a NE and immunogen). For
example, in some embodiments, suitable adjuvants include an
aluminum salt such as aluminum hydroxide gel (alum) or aluminum
phosphate. In some embodiments, an adjuvant may be a salt of
calcium, iron or zinc, or may be an insoluble suspension of
acylated tyrosine, or acylated sugars, cationically or anionically
derivatised polysaccharides, or polyphosphazenes.
[0201] In some embodiments, it is preferred that a composition
comprising a NE and immunogen of the present invention comprises
one or more adjuvants that induce a Th1-type response. However, in
other embodiments, it will be preferred that a composition
comprising a NE and immunogen of the present invention comprises
one or more adjuvants that induce a Th2-type response.
[0202] In general, an immune response is generated to an antigen
through the interaction of the antigen with the cells of the immune
system. Immune responses may be broadly categorized into two
categories: humoral and cell mediated immune responses (e.g.,
traditionally characterized by antibody and cellular effector
mechanisms of protection, respectively). These categories of
response have been termed Th1-type responses (cell-mediated
response), and Th2-type immune responses (humoral response).
[0203] Stimulation of an immune response can result from a direct
or indirect response of a cell or component of the immune system to
an intervention (e.g., exposure to an immunogen). Immune responses
can be measured in many ways including activation, proliferation or
differentiation of cells of the immune system (e.g., B cells, T
cells, dendritic cells, APCs, macrophages, NK cells, NKT cells
etc.); up-regulated or down-regulated expression of markers and
cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly
(including increased spleen cellularity); hyperplasia and mixed
cellular infiltrates in various organs. Other responses, cells, and
components of the immune system that can be assessed with respect
to immune stimulation are known in the art.
[0204] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
compositions and methods of the present invention induce expression
and secretion of cytokines (e.g., by macrophages, dendritic cells
and CD4+ T cells). Modulation of expression of a particular
cytokine can occur locally or systemically. It is known that
cytokine profiles can determine T cell regulatory and effector
functions in immune responses. In some embodiments, Th1-type
cytokines can be induced, and thus, the immunostimulatory
compositions of the present invention can promote a Th1 type
antigen-specific immune response including cytotoxic T-cells (e.g.,
thereby avoiding unwanted Th2 type immune responses (e.g.,
generation of Th2 type cytokines (e.g., IL-13) involved in
enhancing the severity of disease (e.g., IL-13 induction of mucus
formation))).
[0205] Cytokines play a role in directing the T cell response.
Helper (CD4+) T cells orchestrate the immune response of mammals
through production of soluble factors that act on other immune
system cells, including B and other T cells. Most mature CD4+ T
helper cells express one of two cytokine profiles: Th1 or Th2.
Th1-type CD4+ T cells secrete IL-2, IL-3, IFN-.gamma., GM-CSF and
high levels of TNF-.alpha.. Th2 cells express IL-3, IL-4, IL-5,
IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-.alpha.. Th1
type cytokines promote both cell-mediated immunity, and humoral
immunity that is characterized by immunoglobulin class switching to
IgG2a in mice and IgG1 in humans. Th1 responses may also be
associated with delayed-type hypersensitivity and autoimmune
disease. Th2 type cytokines induce primarily humoral immunity and
induce class switching to IgG1 and IgE. The antibody isotypes
associated with Th1 responses generally have neutralizing and
opsonizing capabilities whereas those associated with Th2 responses
are associated more with allergic responses.
[0206] Several factors have been shown to influence skewing of an
immune response towards either a Th1 or Th2 type response. The best
characterized regulators are cytokines IL-12 and IFN-.gamma. are
positive Th1 and negative Th2 regulators. IL-12 promotes
IFN-.gamma. production, and IFN-.gamma. provides positive feedback
for IL-12. IL-4 and IL-10 appear important for the establishment of
the Th2 cytokine profile and to down-regulate Th1 cytokine
production.
[0207] Thus, in preferred embodiments, the present invention
provides a method of stimulating a Th1-type immune response in a
subject comprising administering to a subject a composition
comprising a NE and an immunogen. However, in other embodiments,
the present invention provides a method of stimulating a Th2-type
immune response in a subject (e.g., if balancing of a T cell
mediated response is desired) comprising administering to a subject
a composition comprising a NE and an immunogen. In further
preferred embodiments, adjuvants can be used (e.g., can be
co-administered with a composition of the present invention) to
skew an immune response toward either a Th1 or Th2 type immune
response. For example, adjuvants that induce Th2 or weak Th1
responses include, but are not limited to, alum, saponins, and
SB-As4. Adjuvants that induce Th1 responses include but are not
limited to MPL, MDP, ISCOMS, IL-12, IFN-.gamma., and SB-AS2.
[0208] Several other types of Th1-type immunogens can be used
(e.g., as an adjuvant) in compositions and methods of the present
invention. These include, but are not limited to, the following. In
some embodiments, monophosphoryl lipid A (e.g., in particular
3-de-O-acylated monophosphoryl lipid A (3D-MPL)), is used. 3D-MPL
is a well known adjuvant manufactured by Ribi Immunochem, Montana.
Chemically it is often supplied as a mixture of 3-de-O-acylated
monophosphoryl lipid A with either 4, 5, or 6 acylated chains. In
some embodiments, diphosphoryl lipid A, and 3-O-deacylated variants
thereof are used. Each of these immunogens can be purified and
prepared by methods described in GB 2122204B, hereby incorporated
by reference in its entirety. Other purified and synthetic
lipopolysaccharides have been described (See, e.g., U.S. Pat. No.
6,005,099 and EP 0 729 473; Hilgers et al., 1986, Int. Arch.
Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987, Immunology,
60(1):141-6; and EP 0 549 074, each of which is hereby incorporated
by reference in its entirety). In some embodiments, 3D-MPL is used
in the form of a particulate formulation (e.g., having a small
particle size less than 0.2 .mu.m in diameter, described in EP 0
689 454, hereby incorporated by reference in its entirety).
[0209] In some embodiments, saponins are used as an immunogen
(e.g., Th1-type adjuvant) in a composition of the present
invention. Saponins are well known adjuvants (See, e.g.,
Lacaille-Dubois and Wagner (1996) Phytomedicine vol 2 pp 363-386).
Examples of saponins include Quil A (derived from the bark of the
South American tree Quillaja Saponaria Molina), and fractions
thereof (See, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev Ther
Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279, each of
which is hereby incorporated by reference in its entirety). Also
contemplated to be useful in the present invention are the
haemolytic saponins QS7, QS 17, and QS21 (HPLC purified fractions
of Quil A; See, e.g., Kensil et al. (1991). J. Immunology 146,
431-437, U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0
362 279, each of which is hereby incorporated by reference in its
entirety). Also contemplated to be useful are combinations of QS21
and polysorbate or cyclodextrin (See, e.g., WO 99/10008, hereby
incorporated by reference in its entirety.
[0210] In some embodiments, an immunogenic oligonucleotide
containing unmethylated CpG dinucleotides ("CpG") is used as an
adjuvant in the present invention. CpG is an abbreviation for
cytosine-guanosine dinucleotide motifs present in DNA. CpG is known
in the art as being an adjuvant when administered by both systemic
and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis et
al., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.
Immunol., 1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660,
each of which is hereby incorporated by reference in its entirety).
For example, in some embodiments, the immunostimulatory sequence is
Purine-Purine-C-G-pyrimidine-pyrimidine; wherein the CG motif is
not methylated.
[0211] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
the presence of one or more CpG oligonucleotides activate various
immune subsets including natural killer cells (which produce
IFN-.gamma.) and macrophages. In some embodiments, CpG
oligonucleotides are formulated into a composition of the present
invention for inducing an immune response. In some embodiments, a
free solution of CpG is co-administered together with an antigen
(e.g., present within a NE solution (See, e.g., WO 96/02555; hereby
incorporated by reference). In some embodiments, a CpG
oligonucleotide is covalently conjugated to an antigen (See, e.g.,
WO 98/16247, hereby incorporated by reference), or formulated with
a carrier such as aluminium hydroxide (See, e.g., Brazolot-Millan
et al., Proc. Natl. Acad Sci., USA, 1998, 95(26), 15553-8).
[0212] In some embodiments, adjuvants such as Complete Freunds
Adjuvant and Incomplete Freunds Adjuvant, cytokines (e.g.,
interleukins (e.g., IL-2, IFN-.gamma., IL-4, etc.), macrophage
colony stimulating factor, tumor necrosis factor, etc.), 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., WO93/13202 and WO92/19265, each of which
is hereby incorporated by reference), and other immunogenic
substances (e.g., that enhance the effectiveness of a composition
of the present invention) are used with a composition comprising a
NE and immunogen of the present invention.
[0213] Additional examples of adjuvants that find use in the
present invention include poly(di(carboxylatophenoxy)phosphazene
(PCPP polymer; Virus Research Institute, USA); derivatives of
lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi
ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide
(MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); 0M-174 (a
glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin,
Switzerland); and Leishmania elongation factor (a purified
Leishmania protein; Corixa Corporation, Seattle, Wash.).
[0214] Adjuvants may be added to a composition comprising a NE and
an immunogen, or, the adjuvant may be formulated with carriers, for
example liposomes, or metallic salts (e.g., aluminium salts (e.g.,
aluminium hydroxide)) prior to combining with or co-administration
with a composition comprising a NE and an immunogen.
[0215] In some embodiments, a composition comprising a NE and an
immunogen comprises a single adjuvant. In other embodiments, a
composition comprising a NE and an immunogen comprises two or more
adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO
98/56414; WO 99/12565; WO 99/11241; and WO 94/00153, each of which
is hereby incorporated by reference in its entirety).
[0216] In some embodiments, a composition comprising a NE and an
immunogen of the present invention comprises one or more
mucoadhesives (See, e.g., U.S. Pat. App. No. 20050281843, hereby
incorporated by reference in its entirety). The present invention
is not limited by the type of mucoadhesive utilized. Indeed, a
variety of mucoadhesives are contemplated to be useful in the
present invention including, but not limited to, cross-linked
derivatives of poly(acrylic acid) (e.g., carbopol and
polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone,
polysaccharides (e.g., alginate and chitosan), hydroxypropyl
methylcellulose, lectins, fimbrial proteins, and
carboxymethylcellulose. Although an understanding of the mechanism
is not necessary to practice the present invention and the present
invention is not limited to any particular mechanism of action, in
some embodiments, use of a mucoadhesive (e.g., in a composition
comprising a NE and immunogen) enhances induction of an immune
response in a subject (e.g., administered a composition of the
present invention) due to an increase in duration and/or amount of
exposure to an immunogen that a subject experiences when a
mucoadhesive is used compared to the duration and/or amount of
exposure to an immunogen in the absence of using the
mucoadhesive.
[0217] In some embodiments, a composition of the present invention
may comprise sterile aqueous preparations. Acceptable vehicles and
solvents include, but are not limited to, water, Ringer's solution,
phosphate buffered saline and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed
mineral or non-mineral oil may be employed including synthetic
mono-ordi-glycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables. Carrier formulations
suitable for mucosal, subcutaneous, intramuscular, intraperitoneal,
intravenous, or administration via other routes may be found in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa.
[0218] A composition comprising a NE and an immunogen of the
present invention can be used therapeutically (e.g., to enhance an
immune response) or as a prophylactic (e.g., for immunization
(e.g., to prevent signs or symptoms of disease)). A composition
comprising a NE and an immunogen of the present invention can be
administered to a subject via a number of different delivery routes
and methods.
[0219] For example, the compositions of the present invention can
be administered to a subject (e.g., mucosally (e.g., nasal mucosa,
vaginal mucosa, etc.)) by multiple methods, including, but not
limited to: being suspended in a solution and applied to a surface;
being suspended in a solution and sprayed onto a surface using a
spray applicator; being mixed with a mucoadhesive and applied
(e.g., sprayed or wiped) onto a surface (e.g., mucosal surface);
being placed on or impregnated onto a nasal and/or vaginal
applicator and applied; being applied by a controlled-release
mechanism; being applied as a liposome; or being applied on a
polymer.
[0220] In some preferred embodiments, compositions of the present
invention are administered mucosally (e.g., using standard
techniques; See, e.g., Remington: The Science and Practice of
Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995
(e.g., for mucosal delivery techniques, including intranasal,
pulmonary, vaginal and rectal techniques), as well as European
Publication No. 517,565 and Illum et al., J. Controlled Rel., 1994,
29:133-141 (e.g., for techniques of intranasal administration),
each of which is hereby incorporated by reference in its entirety).
Alternatively, the compositions of the present invention may be
administered dermally or transdermally, using standard techniques
(See, e.g., Remington: The Science arid Practice of Pharmacy, Mack
Publishing Company, Easton, Pa., 19th edition, 1995). The present
invention is not limited by the route of administration.
[0221] Although an understanding of the mechanism is not necessary
to practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
mucosal vaccination is the preferred route of administration as it
has been shown that mucosal administration of antigens has a
greater efficacy of inducing protective immune responses at mucosal
surfaces (e.g., mucosal immunity), the route of entry of many
pathogens. In addition, mucosal vaccination, such as intranasal
vaccination, may induce mucosal immunity not only in the nasal
mucosa, but also in distant mucosal sites such as the genital
mucosa (See, e.g., Mestecky, Journal of Clinical Immunology,
7:265-276, 1987). More advantageously, in further preferred
embodiments, in addition to inducing mucosal immune responses,
mucosal vaccination also induces systemic immunity. In some
embodiments, non-parenteral administration (e.g., mucosal
administration of vaccines) provides an efficient and convenient
way to boost systemic immunity (e.g., induced by parenteral or
mucosal vaccination (e.g., in cases where multiple boosts are used
to sustain a vigorous systemic immunity)).
[0222] In some embodiments, a composition comprising a NE and an
immunogen of the present invention may be used to protect or treat
a subject susceptible to, or suffering from, disease by means of
administering a composition of the present invention via a mucosal
route (e.g., an oral/alimentary or nasal route). Alternative
mucosal routes include intravaginal and intra-rectal routes. In
preferred embodiments of the present invention, a nasal route of
administration is used, termed "intranasal administration" or
"intranasal vaccination" herein. Methods of intranasal vaccination
are well known in the art, including the administration of a
droplet or spray form of the vaccine into the nasopharynx of a
subject to be immunized. In some embodiments, a nebulized or
aerosolized composition comprising a NE and immunogen is provided.
Enteric formulations such as gastro resistant capsules for oral
administration, suppositories for rectal or vaginal administration
also form part of this invention. Compositions of the present
invention may also be administered via the oral route. Under these
circumstances, a composition comprising a NE and an immunogen may
comprise a pharmaceutically acceptable excipient and/or include
alkaline buffers, or enteric capsules. Formulations for nasal
delivery may include those with dextran or cyclodextran and saponin
as an adjuvant.
[0223] Compositions of the present invention may also be
administered via a vaginal route. In such cases, a composition
comprising a NE and an immunogen may comprise pharmaceutically
acceptable excipients and/or emulsifiers, polymers (e.g.,
CARBOPOL), and other known stabilizers of vaginal creams and
suppositories. In some embodiments, compositions of the present
invention are administered via a rectal route. In such cases, a
composition comprising a NE and an immunogen may comprise
excipients and/or waxes and polymers known in the art for forming
rectal suppositories.
[0224] In some embodiments, the same route of administration (e.g.,
mucosal administration) is chosen for both a priming and boosting
vaccination. In some embodiments, multiple routes of administration
are utilized (e.g., at the same time, or, alternatively,
sequentially) in order to stimulate an immune response (e.g., using
a composition comprising a NE and immunogen of the present
invention).
[0225] For example, in some embodiments, a composition comprising a
NE and an immunogen is administered to a mucosal surface of a
subject in either a priming or boosting vaccination regime.
Alternatively, in some embodiments, a composition comprising a NE
and an immunogen is administered systemically in either a priming
or boosting vaccination regime. In some embodiments, a composition
comprising a NE and an immunogen is administered to a subject in a
priming vaccination regimen via mucosal administration and a
boosting regimen via systemic administration. In some embodiments,
a composition comprising a NE and an immunogen is administered to a
subject in a priming vaccination regimen via systemic
administration and a boosting regimen via mucosal administration.
Examples of systemic routes of administration include, but are not
limited to, a parenteral, intramuscular, intradermal, transdermal,
subcutaneous, intraperitoneal or intravenous administration. A
composition comprising a NE and an immunogen may be used for both
prophylactic and therapeutic purposes.
[0226] In some embodiments, compositions of the present invention
are administered by pulmonary delivery. For example, a composition
of the present invention can be delivered to the lungs of a subject
(e.g., a human) via inhalation (e.g., thereby traversing across the
lung epithelial lining to the blood stream (See, e.g., Adjei, et
al. Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J.
Pharmaceutics 1990; 63:135-144; Braquet, et al. J. Cardiovascular
Pharmacology 1989 143-146; Hubbard, et al. (1989) Annals of
Internal Medicine, Vol. III, pp. 206-212; Smith, et al. J. Clin.
Invest. 1989; 84:1145-1146; Oswein, et al. "Aerosolization of
Proteins", 1990; Proceedings of Symposium on Respiratory Drug
Delivery II Keystone, Colo.; Debs, et al. J. Immunol. 1988;
140:3482-3488; and U.S. Pat. No. 5,284,656 to Platz, et al, each of
which are hereby incorporated by reference in its entirety). A
method and composition for pulmonary delivery of drugs for systemic
effect is described in U.S. Pat. No. 5,451,569 to Wong, et al.,
hereby incorporated by reference; See also U.S. Pat. No. 6,651,655
to Licalsi et al., hereby incorporated by reference in its
entirety)).
[0227] Further contemplated for use in the practice of this
invention are a wide range of mechanical devices designed for
pulmonary and/or nasal mucosal delivery of pharmaceutical agents
including, but not limited to, nebulizers, metered dose inhalers,
and powder inhalers, all of which are familiar to those skilled in
the art. Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II
nebulizer (Marquest Medical Products, Englewood, Colo.); the
Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park,
N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford,
Mass.). All such devices require the use of formulations suitable
for dispensing of the therapeutic agent. Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants, surfactants, carriers and/or
other agents useful in therapy. Also, the use of liposomes,
microcapsules or microspheres, inclusion complexes, or other types
of carriers is contemplated.
[0228] Thus, in some embodiments, a composition comprising a NE and
an immunogen of the present invention may be used to protect and/or
treat a subject susceptible to, or suffering from, a disease by
means of administering a compositions comprising a NE and an
immunogen by mucosal, intramuscular, intraperitoneal, intradermal,
transdermal, pulmonary, intravenous, subcutaneous or other route of
administration described herein. Methods of systemic administration
of the vaccine preparations may include conventional syringes and
needles, or devices designed for ballistic delivery of solid
vaccines (See, e.g., WO 99/27961, hereby incorporated by
reference), or needleless pressure liquid jet device (See, e.g.,
U.S. Pat. No. 4,596,556; U.S. Pat. No. 5,993,412, each of which are
hereby incorporated by reference), or transdermal patches (See,
e.g., WO 97/48440; WO 98/28037, each of which are hereby
incorporated by reference). The present invention may also be used
to enhance the immunogenicity of antigens applied to the skin
(transdermal or transcutaneous delivery, See, e.g., WO 98/20734; WO
98/28037, each of which are hereby incorporated by reference).
Thus, in some embodiments, the present invention provides a
delivery device for systemic administration, pre-filled with the
vaccine composition of the present invention.
[0229] The present invention is not limited by the type of subject
administered (e.g., in order to stimulate an immune response (e.g.,
in order to generate protective immunity (e.g., mucosal and/or
systemic immunity))) a composition of the present invention.
Indeed, a wide variety of subjects are contemplated to be benefited
from administration of a composition of the present invention. In
preferred embodiments, the subject is a human. In some embodiments,
human subjects are of any age (e.g., adults, children, infants,
etc.) that have been or are likely to become exposed to a
microorganism (e.g., Streptococcal bacteria (e.g., Streptococcus
pneumoniae)). In some embodiments, the human subjects are subjects
that are more likely to receive a direct exposure to pathogenic
microorganisms or that are more likely to display signs and
symptoms of disease after exposure to a pathogen (e.g., immune
suppressed subjects). In some embodiments, the general public is
administered (e.g., vaccinated with) a composition of the present
invention (e.g., to prevent the occurrence or spread of disease).
For example, in some embodiments, compositions and methods of the
present invention are utilized to vaccinate a group of people
(e.g., a population of a region, city, state and/or country) for
their own health (e.g., to prevent or treat disease). In some
embodiments, the subjects are non-human mammals (e.g., pigs,
cattle, goats, horses, sheep, or other livestock; or mice, rats,
rabbits or other animal). In some embodiments, compositions and
methods of the present invention are utilized in research settings
(e.g., with research animals). In some embodiments, the present
invention provides a method to elicit an immune response (e.g.,
protective immune response) in infants (e.g., from about 0-2 years
old) by administering to the infant a safe and effective amount of
an immunogenic composition of the invention (e.g., a pediatric
vaccine). Further embodiments of the invention include the
provision of the immunogenic S. pneumoniae compositions of the
invention for use in medicine and the use of the S. pneumoniae
compositions of the invention in the manufacture of a medicament
for the prevention (or treatment) of pneumococcal disease.
[0230] In yet another embodiment, the present invention is provides
a method to elicit an immune response (e.g., a protective immune
response) in the elderly population (e.g., in a subject 50 years or
over in age, typically over 55 years and more generally over 60
years) by administering a safe and effective amount of an
immunogenic composition of the invention.
[0231] A composition of the present invention may be formulated for
administration by any route, such as mucosal, oral, topical,
parenteral or other route described herein. The compositions may be
in any one or more different forms including, but not limited to,
tablets, capsules, powders, granules, lozenges, foams, creams or
liquid preparations.
[0232] Topical formulations of the present invention may be
presented as, for instance, ointments, creams or lotions, foams,
and aerosols, and may contain appropriate conventional additives
such as preservatives, solvents (e.g., to assist penetration), and
emollients in ointments and creams.
[0233] Topical formulations may also include agents that enhance
penetration of the active ingredients through the skin. Exemplary
agents include a binary combination of N-(hydroxyethyl) pyrrolidone
and a cell-envelope disordering compound, a sugar ester in
combination with a sulfoxide or phosphine oxide, and sucrose
monooleate, decyl methyl sulfoxide, and alcohol.
[0234] Other exemplary materials that increase skin penetration
include surfactants or wetting agents including, but not limited
to, polyoxyethylene sorbitan mono-oleoate (Polysorbate 80);
sorbitan mono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol
polymer (Triton WR-1330); polyoxyethylene sorbitan tri-oleate
(Tween 85); dioctyl sodium sulfosuccinate; and sodium sarcosinate
(Sarcosyl NL-97); and other pharmaceutically acceptable
surfactants.
[0235] In certain embodiments of the invention, compositions may
further comprise one or more alcohols, zinc-containing compounds,
emollients, humectants, thickening and/or gelling agents,
neutralizing agents, and surfactants. Water used in the
formulations is preferably deionized water having a neutral pH.
Additional additives in the topical formulations include, but are
not limited to, silicone fluids, dyes, fragrances, pH adjusters,
and vitamins.
[0236] Topical formulations may also contain compatible
conventional carriers, such as cream or ointment bases and ethanol
or oleyl alcohol for lotions. Such carriers may be present as from
about 1% up to about 98% of the formulation. The ointment base can
comprise one or more of petrolatum, mineral oil, ceresin, lanolin
alcohol, panthenol, glycerin, bisabolol, cocoa butter and the
like.
[0237] In some embodiments, pharmaceutical compositions of the
present invention may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product.
[0238] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions. Thus, for example, the compositions
may contain additional, compatible, pharmaceutically-active
materials such as, for example, antipuritics, astringents, local
anesthetics or anti-inflammatory agents, or may contain additional
materials useful in physically formulating various dosage forms of
the compositions of the present invention, such as dyes, flavoring
agents, preservatives, antioxidants, opacifiers, thickening agents
and stabilizers. However, such materials, when added, preferably do
not unduly interfere with the biological activities of the
components of the compositions of the present invention. The
formulations can be sterilized and, if desired, mixed with
auxiliary agents (e.g., lubricants, preservatives, stabilizers,
wetting agents, emulsifiers, salts for influencing osmotic
pressure, buffers, colorings, flavorings and/or aromatic substances
and the like) that do not deleteriously interact with the NE and
immunogen of the formulation. In some embodiments,
immunostimulatory compositions of the present invention are
administered in the form of a pharmaceutically acceptable salt.
When used the salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically acceptable salts thereof. Such salts
include, but are not limited to, those prepared from the following
acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric,
maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric,
methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0239] Suitable buffering agents include, but are not limited to,
acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3%
w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and
a salt (0.8-2% w/v). Suitable preservatives may include
benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%
w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02%
w/v).
[0240] In some embodiments, a composition comprising a NE and an
immunogen is co-administered with one or more antibiotics. For
example, one or more antibiotics may be administered with, before
and/or after administration of a composition comprising a NE and an
immunogen. The present invention is not limited by the type of
antibiotic co-administered. Indeed, a variety of antibiotics may be
co-administered including, but not limited to, .beta.-lactam
antibiotics, penicillins (such as natural penicillins,
aminopenicillins, penicillinase-resistant penicillins, carboxy
penicillins, ureido penicillins), cephalosporins (first generation,
second generation, and third generation cephalosporins), and other
.beta.-lactams (such as imipenem, monobactams,), .beta.-lactamase
inhibitors, vancomycin, aminoglycosides and spectinomycin,
tetracyclines, chloramphenicol, erythromycin, lincomycin,
clindamycin, rifampin, metronidazole, polymyxins, doxycycline,
quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and
quinolines.
[0241] There are an enormous amount of antimicrobial agents
currently available for use in treating bacterial, fungal and viral
infections. For a comprehensive treatise on the general classes of
such drugs and their mechanisms of action, the skilled artisan is
referred to Goodman & Gilman's "The Pharmacological Basis of
Therapeutics" Eds. Hardman et al., 9th Edition, Pub. McGraw Hill,
chapters 43 through 50, 1996, (herein incorporated by reference in
its entirety). Generally, these agents include agents that inhibit
cell wall synthesis (e.g., penicillins, cephalosporins,
cycloserine, vancomycin, bacitracin); and the imidazole antifungal
agents (e.g., miconazole, ketoconazole and clotrimazole); agents
that act directly to disrupt the cell membrane of the microorganism
(e.g., detergents such as polmyxin and colistimethate and the
antifungals nystatin and amphotericin B); agents that affect the
ribosomal subunits to inhibit protein synthesis (e.g.,
chloramphenicol, the tetracyclines, erythromycin and clindamycin);
agents that alter protein synthesis and lead to cell death (e.g.,
aminoglycosides); agents that affect nucleic acid metabolism (e.g.,
the rifamycins and the quinolones); the antimetabolites (e.g.,
trimethoprim and sulfonamides); and the nucleic acid analogues such
as zidovudine, gangcyclovir, vidarabine, and acyclovir which act to
inhibit viral enzymes essential for DNA synthesis. Various
combinations of antimicrobials may be employed.
[0242] The present invention also includes methods involving
co-administration of a composition comprising a NE and an immunogen
with one or more additional active and/or immunostimulatory agents
(e.g., a composition comprising a NE and a different immunogen, an
antibiotic, anti-oxidant, etc.). Indeed, it is a further aspect of
this invention to provide methods for enhancing prior art
immunostimulatory methods (e.g., immunization methods) and/or
pharmaceutical compositions by co-administering a composition of
the present invention. In co-administration procedures, the agents
may be administered concurrently or sequentially. In one
embodiment, the compositions described herein are administered
prior to the other active agent(s). The pharmaceutical formulations
and modes of administration may be any of those described herein.
In addition, the two or more co-administered agents may each be
administered using different modes (e.g., routes) or different
formulations. The additional agents to be co-administered (e.g.,
antibiotics, adjuvants, etc.) can be any of the well-known agents
in the art, including, but not limited to, those that are currently
in clinical use.
[0243] In some embodiments, a composition comprising a NE and
immunogen is administered to a subject via more than one route. For
example, a subject that would benefit from having a protective
immune response (e.g., immunity) towards a pathogenic microorganism
may benefit from receiving mucosal administration (e.g., nasal
administration or other mucosal routes described herein) and,
additionally, receiving one or more other routes of administration
(e.g., parenteral or pulmonary administration (e.g., via a
nebulizer, inhaler, or other methods described herein). In some
preferred embodiments, administration via mucosal route is
sufficient to induce both mucosal as well as systemic immunity
towards an immunogen or organism from which the immunogen is
derived. In other embodiments, administration via multiple routes
serves to provide both mucosal and systemic immunity. Thus,
although an understanding of the mechanism is not necessary to
practice the present invention and the present invention is not
limited to any particular mechanism of action, in some embodiments,
it is contemplated that a subject administered a composition of the
present invention via multiple routes of administration (e.g.,
immunization (e.g., mucosal as well as airway or parenteral
administration of a composition comprising a NE and immunogen of
the present invention) may have a stronger immune response to an
immunogen than a subject administered a composition via just one
route.
[0244] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compositions, increasing
convenience to the subject and a physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer based systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109,
hereby incorporated by reference. Delivery systems also include
non-polymer systems that are: lipids including sterols such as
cholesterol, cholesterol esters and fatty acids or neutral fats
such as mono-di- and tri-glycerides; hydrogel release systems;
sylastic systems; peptide based systems; wax coatings; compressed
tablets using conventional binders and excipients; partially fused
implants; and the like. Specific examples include, but are not
limited to: (a) erosional systems in which an agent of the
invention is contained in a form within a matrix such as those
described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152,
each of which is hereby incorporated by reference and (b)
diffusional systems in which an active component permeates at a
controlled rate from a polymer such as described in U.S. Pat. Nos.
3,854,480, 5,133,974 and 5,407,686, each of which is hereby
incorporated by reference. In addition, pump-based hardware
delivery systems can be used, some of which are adapted for
implantation.
[0245] In preferred embodiments, a composition comprising a NE and
an immunogen of the present invention comprises a suitable amount
of the immunogen to induce an immune response in a subject when
administered to the subject. In preferred embodiments, the immune
response is sufficient to provide the subject protection (e.g.,
immune protection) against a subsequent exposure to the immunogen
or the microorganism (e.g., Streptococcal bacteria (e.g.,
Streptococcus pneumoniae)) from which the immunogen was derived.
The present invention is not limited by the amount of immunogen
used. In some preferred embodiments, the amount of immunogen (e.g.,
Streptococcal bacteria (e.g., Streptococcus pneumoniae) or one or
more component parts thereof) in a composition comprising a NE and
immunogen (e.g., for use as an immunization dose) is selected as
that amount which induces an immunoprotective response without
significant, adverse side effects. The amount will vary depending
upon which specific immunogen or combination thereof is/are
employed, and can vary from subject to subject, depending on a
number of factors including, but not limited to, the species, age
and general condition (e.g., health) of the subject, and the mode
of administration.
[0246] In some embodiments, each dose (e.g., of a composition
comprising a NE and an immunogen (e.g., administered to a subject
to induce an immune response (e.g., a protective immune response
(e.g., protective immunity))) comprises between about 10.sup.5 and
10.sup.8 colony forming units (CFU) of Streptococcus (e.g.,
Streptococcus pneumoniae) of killed/inactivated bacteria, although
greater (e.g., about 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, or
more) and lesser (e.g., about 10.sup.4, 10.sup.3, 10.sup.2 or
fewer) CFU of Streptococcus (e.g., Streptococcus pneumoniae) (e.g.,
killed whole Streptococcus pneumoniae) may also be utilized. In
some embodiments, a nanoemulsion solution is utilized to inactivate
the Streptococcus (e.g., Streptococcus pneumoniae). In some
embodiments, a nanoemulsion solution is utilized to inactivate the
Streptococcus (e.g., Streptococcus pneumoniae). In some
embodiments, the nanoemulsion comprises W.sub.805EC. In some
embodiments, the immunity protects the subject from displaying
signs or symptoms of disease caused by Streptococcus (e.g.,
Streptococcus pneumoniae). In some embodiments, the immunity
protects the subject from challenge with a subsequent exposure to
live Streptococcus (e.g., Streptococcus pneumoniae). In some
embodiments, each dose (e.g., administered to a subject to induce
and immune response)) comprises between 10 and 10.sup.10 CFU of
Streptococcus (e.g., Streptococcus pneumoniae) per dose. In some
embodiments, each dose comprises between 10.sup.5 and 10.sup.8 CFU
of Streptococcus (e.g., Streptococcus pneumoniae) per dose. In some
embodiments, each dose comprises between 10.sup.3 and 10.sup.5 CFU
of Streptococcus (e.g., Streptococcus pneumoniae) per dose. In some
embodiments, each dose comprises between 10.sup.5 and 10.sup.8 CFU
of Streptococcus (e.g., Streptococcus pneumoniae) per dose; in some
embodiments, each dose comprises 10.sup.5 CFU of Streptococcus
(e.g., Streptococcus pneumoniae) per dose. In some embodiments,
each dose comprises 10.sup.6 CFU of Streptococcus (e.g.,
Streptococcus pneumoniae) per dose. In some embodiments, each dose
comprises 10.sup.7 CFU of Streptococcus (e.g., Streptococcus
pneumoniae) per dose. In some embodiments, each dose comprises more
than 10.sup.8 CFU of Streptococcus (e.g., Streptococcus pneumoniae)
per dose. In some preferred embodiments, each dose comprises
10.sup.8 CFU of Streptococcus (e.g., Streptococcus pneumoniae) per
dose.
[0247] In some embodiments, each dose (e.g., of a composition
comprising a NE and an immunogen (e.g., administered to a subject
to induce an immune response (e.g., a protective immune response
(e.g., protective immunity))) comprises 0.05-5000 .mu.g of an
additional immunogen (e.g., recombinant and/or purified protein,
adjuvant (e.g., cholera toxin), etc.). In some embodiments, each
dose will comprise 1-500 .mu.g, in some embodiments, each dose will
comprise 350-750 .mu.g, in some embodiments, each dose will
comprise 50-200 .mu.g, in some embodiments, each dose will comprise
25-75 .mu.g of immunogen (e.g., recombinant and/or purified
protein). In some embodiments, each dose comprises an amount of the
immunogen sufficient to generate an immune response. An effective
amount of the immunogen in a dose need not be quantified, as long
as the amount of immunogen generates an immune response in a
subject when administered to the subject.
[0248] In some embodiments, it is expected that each dose (e.g., of
a composition comprising a NE and an immunogen (e.g., administered
to a subject to induce and immune response)) is from 0.001 to 15%
or more (e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more)
by weight immunogen (e.g., neutralized bacteria, or recombinant
and/or purified protein). In some embodiments, an initial or prime
administration dose contains more immunogen than a subsequent boost
dose
[0249] In some embodiments, when a NE of the present invention is
utilized to inactivate a live microorganism, each dose (e.g.,
administered to a subject to induce and immune response)) comprises
between 10 and 10.sup.9 CFU of the microorganism per dose; in some
embodiments, each dose comprises between 10.sup.5 and 10.sup.8 CFU
of the microorganism per dose; in some embodiments, each dose
comprises between 10.sup.3 and 10.sup.5 CFU of the microorganism
per dose; in some embodiments, each dose comprises between 10.sup.2
and 10.sup.4 CFU of the microorganism per dose; in some
embodiments, each dose comprises 10 CFU of the microorganism per
dose; in some embodiments, each dose comprises 10.sup.2 CFU of the
microorganism per dose; and in some embodiments, each dose
comprises 10.sup.4 CFU of the microorganism per dose. In some
embodiments, each dose comprises more than 10.sup.9 CFU of the
microorganism per dose. In some preferred embodiments, each dose
comprises 10.sup.3 CFU of the microorganism per dose.
[0250] The present invention is not limited by the amount of NE
used to inactivate live microorganisms (e.g., Streptococcal
bacteria (e.g., S. pneumoniae)). In some embodiments, a 0.1%-5% NE
solution is used, in some embodiments, a 5%-20% NE solution is
used, in some embodiments, a 20% NE solution is used, and in some
embodiments, a NE solution greater than 20% is used order to
inactivate a pathogenic microorganism. In preferred embodiments, a
15% NE solution is used.
[0251] Similarly, the present invention is not limited by the
duration of time a live microorganism is incubated in a NE of the
present invention in order to become inactivated. In some
embodiments, the microorganism is incubated for 1-3 hours in NE. In
some embodiments, the microorganism is incubated for 3-6 hours in
NE. In some embodiments, the microorganism is incubated for more
than 6 hours in NE. In preferred embodiments, the microorganism is
incubated for 3 hours in NE (e.g., a 10% NE solution). In some
embodiments, the incubation is carried out at 37.degree. C. In some
embodiments, the incubation is carried out at a temperature greater
than or less than 37.degree. C. The present invention is also not
limited by the amount of microorganism used for inactivation. The
amount of microorganism may depend upon a number of factors
including, but not limited to, the total amount of immunogenic
composition (e.g., NE and immunogen) desired, the concentration of
solution desired (e.g., prior to dilution for administration), the
microorganism and the NE. In some preferred embodiments, the amount
of microorganism used in an inactivation procedure is that amount
that produces the desired amount of immunogen (e.g., as described
herein) to be administered in a single dose (e.g., diluted from a
concentrated stock) to a subject.
[0252] In some embodiments, a composition comprising a NE and an
immunogen of the present invention is formulated in a concentrated
dose that can be diluted prior to administration to a subject. For
example, dilutions of a concentrated composition may be
administered to a subject such that the subject receives any one or
more of the specific dosages provided herein. In some embodiments,
dilution of a concentrated composition may be made such that a
subject is administered (e.g., in a single dose) a composition
comprising 0.5-50% of the NE and immunogen present in the
concentrated composition. In some preferred embodiments, a subject
is administered in a single dose a composition comprising 1% of the
NE and immunogen present in the concentrated composition.
Concentrated compositions are contemplated to be useful in a
setting in which large numbers of subjects may be administered a
composition of the present invention (e.g., an immunization clinic,
hospital, school, etc.). In some embodiments, a composition
comprising a NE and an immunogen of the present invention (e.g., a
concentrated composition) is stable at room temperature for more
than 1 week, in some embodiments for more than 2 weeks, in some
embodiments for more than 3 weeks, in some embodiments for more
than 4 weeks, in some embodiments for more than 5 weeks, and in
some embodiments for more than 6 weeks.
[0253] Generally, the emulsion compositions of the invention will
comprise at least 0.001% to 100%, preferably 0.01 to 90%, of
emulsion per ml of liquid composition. It is envisioned that the
formulations may comprise about 0.001%, about 0.0025%, about
0.005%, about 0.0075%, about 0.01%, about 0.025%, about 0.05%,
about 0.075%, about 0.1%, about 0.25%, about 0.5%, about 1.0%,
about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%, about 90%, about 95% or about 98% of emulsion per
ml of liquid composition. It should be understood that a range
between any two figures listed above is specifically contemplated
to be encompassed within the metes and bounds of the present
invention. Some variation in dosage will necessarily occur
depending on the condition of the specific pathogen and the subject
being immunized.
[0254] In some embodiments, following an initial administration of
a composition of the present invention (e.g., an initial
vaccination), a subject may receive one or more boost
administrations (e.g., around 2 weeks, around 3 weeks, around 4
weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8
weeks, around 10 weeks, around 3 months, around 4 months, around 6
months, around 9 months, around 1 year, around 2 years, around 3
years, around 5 years, around 10 years) subsequent to a first,
second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth,
and/or more than tenth administration. Although an understanding of
the mechanism is not necessary to practice the present invention
and the present invention is not limited to any particular
mechanism of action, in some embodiments, reintroduction of an
immunogen in a boost dose enables vigorous systemic immunity in a
subject. The boost can be with the same formulation given for the
primary immune response, or can be with a different formulation
that contains the immunogen. The dosage regimen will also, at least
in part, be determined by the need of the subject and be dependent
on the judgment of a practitioner.
[0255] Dosage units may be proportionately increased or decreased
based on several factors including, but not limited to, the weight,
age, and health status of the subject. In addition, dosage units
may be increased or decreased for subsequent administrations (e.g.,
boost administrations).
[0256] It is contemplated that the compositions and methods of the
present invention will find use in various settings, including
research settings. For example, compositions and methods of the
present invention also find use in studies of the immune system
(e.g., characterization of adaptive immune responses (e.g.,
protective immune responses (e.g., mucosal or systemic immunity))).
Uses of the compositions and methods provided by the present
invention encompass human and non-human subjects and samples from
those subjects, and also encompass research applications using
these subjects. Compositions and methods of the present invention
are also useful in studying and optimizing nanoemulsions,
immunogens, and other components and for screening for new
components. Thus, it is not intended that the present invention be
limited to any particular subject and/or application setting.
[0257] The formulations can be tested in vivo in a number of animal
models developed for the study of mucosal and other routes of
delivery. As is readily apparent, the compositions of the present
invention are useful for preventing and/or treating a wide variety
of diseases and infections caused by viruses, bacteria, parasites,
and fungi, as well as for eliciting an immune response against a
variety of antigens. Not only can the compositions be used
prophylactically or therapeutically, as described above, the
compositions can also be used in order to prepare antibodies, both
polyclonal and monoclonal (e.g., for diagnostic purposes), as well
as for immunopurification of an antigen of interest. If polyclonal
antibodies are desired, a selected mammal, (e.g., mouse, rabbit,
goat, horse, etc.) can be immunized with the compositions of the
present invention. The animal is usually boosted 2-6 weeks later
with one or more--administrations of the antigen. Polyclonal
antisera can then be obtained from the immunized animal and used
according to known procedures (See, e.g., Jurgens et al., J. Chrom.
1985, 348:363-370).
[0258] In some embodiments, the present invention provides a kit
comprising a composition comprising a NE and an immunogen. In some
embodiments, the kit further provides a device for administering
the composition. The present invention is not limited by the type
of device included in the kit. In some embodiments, the device is
configured for nasal application of the composition of the present
invention (e.g., a nasal applicator (e.g., a syringe) or nasal
inhaler or nasal mister). In some embodiments, a kit comprises a
composition comprising a NE and an immunogen in a concentrated form
(e.g., that can be diluted prior to administration to a
subject).
[0259] In some embodiments, all kit components are present within a
single container (e.g., vial or tube). In some embodiments, each
kit component is located in a single container (e.g., vial or
tube). In some embodiments, one or more kit component are located
in a single container (e.g., vial or tube) with other components of
the same kit being located in a separate container (e.g., vial or
tube). In some embodiments, a kit comprises a buffer. In some
embodiments, the kit further comprises instructions for use.
EXAMPLES
[0260] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0261] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); .mu. (micron); M (Molar);
.mu.M (micromolar); mM (millimolar); N (Normal); mol (moles); mmol
(millimoles); pmol (micromoles); nmol (nanomoles); g (grams); mg
(milligrams); .mu.g (micrograms); ng (nanograms); L (liters); ml
(milliliters); .mu.l (microliters); cm (centimeters); mm
(millimeters); .mu.m (micrometers); nM (nanomolar); .degree. C.
(degrees Centigrade); and PBS (phosphate buffered saline).
Example 1
Immunogenic Streptococcus pneumoniae Compositions
Experimental Design and Materials and Methods.
[0262] Outbred CD-1 or C57/B6 (8 groups; 6 mice per group) were
intranasally immunized with 7.5 .mu.L or 0.14 WCPAg in 1, 5, 10 and
20% NE. WCPAg was generated as follows: strain RX1, a
capsule-negative mutant derived from a pneumococcus capsular
serotype 2 (e.g., an autolysin (lytA)-negative mutant of Rx1
(Rx1AL.sup.-)) grown at 37.degree. C. in Todd-Hewitt broth
supplemented with 0.5% yeast extract (THY) and 0.3 .mu.g of
erythromycin/ml to about 10.sup.9 cells/ml, washed and suspended in
saline at 10% of the original volume, and then mixed 3:7
(volume/volume) with ethanol, washed and resuspended in saline, and
then frozen for later use.
[0263] The mice were given a volume of 12 .mu.L (6 .mu.L per nare)
delivered manually into the nasal cavity of the mouse. Control
groups were: 7.5 .mu.L WCPAg in PBS, cholera toxin (CT), or Alum
(delivered intramuscularly); 0.14 WCPAg in PBS; 20% NE alone.
[0264] The study design is presented in table 1.
TABLE-US-00002 TABLE 1 Design for S. pneumoniae Efficacy Treatment
Conc. WCPAg Cells/ Volume N Day 0, Week 4, Week 8 (cells/ml)
inoculum (.mu.l) (CD1) 7.5 .mu.l WCPAg + 1% NE 1.3 .times.
10{circumflex over ( )}10 10{circumflex over ( )}8 12 6 7.5 .mu.l
WCPAg + 5% NE 1.3 .times. 10{circumflex over ( )}10 10{circumflex
over ( )}8 12 6 7.5 .mu.l WCPAg + 10% NE 1.3 .times. 10{circumflex
over ( )}10 10{circumflex over ( )}8 12 6 7.5 .mu.l WCPAg + 20% NE
1.3 .times. 10{circumflex over ( )}10 10{circumflex over ( )}8 12 6
0.1 .mu.l WCPAg + 1% NE 1 .times. 10{circumflex over ( )}10
10{circumflex over ( )}6 12 6 0.1 .mu.l WCPAg + 5% NE 1 .times.
10{circumflex over ( )}10 10{circumflex over ( )}6 12 6 0.1 .mu.l
WCPAg + 10% NE 1 .times. 10{circumflex over ( )}10 10{circumflex
over ( )}6 12 6 0.1 .mu.l WCPAg + 20% NE 1 .times. 10{circumflex
over ( )}10 10{circumflex over ( )}6 12 6 7.5 .mu.l WCPAg + 1xPBS
1.3 .times. 10{circumflex over ( )}7 10{circumflex over ( )}8 12 8
20% NE 12 4 7.5 .mu.l WCPAg + Alum IM 1.3 .times. 10{circumflex
over ( )}7 10{circumflex over ( )}8 12 2 7.5 ml WCPAg + CT 1.3
.times. 10{circumflex over ( )}7 10{circumflex over ( )}8 12 4 0.1
.mu.l WCPAg + 1xPBS 1 .times. 10{circumflex over ( )}10
10{circumflex over ( )}6 12 8
[0265] Mice were vaccinated at week 0, 4 and 8 (See, e.g., Malley,
et al. (2005) Proc Natl Acad Sci USA. March 29; 102(13):4848-53).
Blood samples were obtained via saphenous vein bleed at weeks 0, 2,
4, 6, 8, and 10. IgG antibodies against the lysed whole bacterium
were assayed via ELISA after each bleed week.
[0266] At week 12, mice were given rifampin subcutaneously to clear
any nasal colonization. Seven days post rifampin, mice were
challenged with 1.times.10.sup.5 CFU live S. pneumoniae/mouse and
monitored daily for weight and temperature. On day 7 after
challenge, the mice were sacrificed and cardiac bleed was taken for
CBC and IgG titers, heads were preserved for histology and
retrograde nasophyringeal wash was taken for S. pneumoniae colony
counts on blood-agar plates. IgG end-titer ELISA was performed
in-house using standard assays known in the art. CBC was performed
at the University of Michigan Unit for Laboratory Animal Medicine
(ULAM) per standard procedures known in the art
[0267] Test Formulation. Vaccine formulations were prepared by
vigorously mixing WCPAg and NE or Alum or CT in 10.times.PBS and
sterile water for injection for about 20 seconds just prior to
immunization (30 to 60 minutes) (See FIG. 1 for formulations).
[0268] Immunization Procedures. Mice were vaccinated intranasally
(I.N.) or intramuscularly (I.M.) with 3 administrations of vaccine
and antibody responses were measured at two week intervals over a
period of 12 weeks. For I.N. immunization, animals were
anaesthetized with Isoflurane (IMPAC 6) and held in an inverted
position until 6 .mu.L of vaccine, delivered with a pipette tip,
were completely inhaled. For I.M. vaccination, mice were
anaesthetized with Isoflurane (IMPAC 6) and 12 .mu.L of WCPAg/Alum
vaccine was injected into the epaxial muscle.
[0269] Blood Sample Collection. Blood samples were obtained from
the saphenous vein at various time points during the course of the
trials. The final sample was obtained by cardiac puncture from
euthanized, premorbid mice. Serum was separated from the blood by
centrifugation at 1500.times.g for 5 minutes after coagulation.
Serum was stored at -20.degree. C. until used for ELISA. For CBC,
blood was collected after cardiac puncture and placed into
heparinized tubes with continuous rocking until delivery to
ULAM.
[0270] Weight/Temperature Monitoring. Weight and body core
temperature (rectal thermometer BAT-12) temperatures were taken 5-7
days after challenge.
[0271] Rifampin Administration. Two weeks following the last
immunization, animals in all groups received 1 mg of rifampin
subcutaneously on two consecutive days to eliminate pneumococcal
colonization.
[0272] Retrograde Nasopharyngeal Wash. Nasopharyngeal wash was
obtained from mice euthanized by Isoflurane inhalation. After the
trachea was dissected, a 22-gauge catheter (Angiocath, B-D)
attached to a 1 ml syringe was inserted into the trachea further to
the nasal cavity. The nares of the mice were washed with 1 .mu.L
PBS and solution was let to fall onto a blood agar plate coated
with gentamycin.
[0273] Analysis of IgG titers: ELISA for anti-S. pneumoniae
antibodies was utilized to determine IgG titers. Preparation of
WCPAg-coated ELISA Plates. Coating buffer, 0.05M
carbonate-bicarbonate buffer (pH 9.6), was made from
carbonate-bicarbonate buffer capsules (SIGMA) by dissolving the in
double distilled water according to manufacturer's instructions. S.
pneumoniae lysate (whole bacterium lysed in non-ionic lysis buffer)
was diluted in coating buffer and 1004 per well was added to
96-well plates (Nunc, Maxisorb Plates). Plates were incubated
overnight and kept at 4.degree. C. until used. Stored plates were
warmed up 1/2 hour at 37.degree. C. and antigen solution was
removed by inversion and tamping on paper towels. The plates were
blocked with 1% milk in PBS (1004, per well) and incubated at 1
hour at 37.degree. C. Blocking solution was removed from wells just
before diluted serum was added.
[0274] Preparation of the primary antibody dilutions. Mouse sera
was diluted in 0.1% BSA in PBS. 100 .mu.L of the diluted serum was
added per well in duplicate and incubated overnight at 4.degree. C.
The next day, the plates were warmed for 1/2 hour at 37.degree. C.
The serum dilutions were removed by inversion/tamping and washed
3.times. with ELISA wash buffer (Quantikine, R&D Systems).
[0275] Preparation of a recommended dilution of secondary antibody
conjugate. Secondary antibody (goat anti-mouse F(c)
alkaline-phosphatase conjugated (Rockland) was diluted at 1:1000 in
0.1% BSA in PBS. 100 .mu.L of the diluted secondary antibody was
added to each well and incubated 1 hour at 37.degree. C. The
secondary antibody was removed by inversion/tamping and the plate
washed 3 times with ELISA wash buffer (Quantikine, R&D
Systems).
[0276] Preparation of alkaline phosphatase substrate solution.
SigmaFast p-Nitrophenyl Phosphate Tablets (pNPP, SIGMA) were
dissolved in double distilled water according to the manufacturer's
recommendations. After removing the last wash, 100 .mu.L of pNPP
solution was added to each well incubated and read every 1/2 hour
until saturation was achieved. The timepoint closest to saturation
was chosen as analysis time.
[0277] End-point determination. The antibody endpoint titers are
defined as the reciprocal of the highest serum dilution which gives
a reading above cutoff value determined by the dilution of control
sera and plate background (passes at least two standard deviations
above average for background wells, See, e.g., Frey, et al. (1998)
Journal of Immunological Methods 221:35-41; Classen, et al. (1987)
Journal of Clinical Microbiology. 25:600).
Example 2
Distribution of Anti-S. pneumoniae Antibody Titers at 8 Weeks after
Two Vaccinations
[0278] FIG. 2 shows anti-S. pneumoniae IgG titer distributions at
week 8, after two intranasal vaccine doses given one month apart on
weeks 0 and 4, for experimental and control groups. All mice
vaccinated with 10.sup.8 CFU WCPAg plus varying concentrations of
nanoemulsion obtained a serum antibody titer of 5.times.10.sup.3 or
greater, with a maximum of 10.sup.5 and an average across all
groups of 7.times.10.sup.5. Titers for individual animals (circles)
and mean serum titer per group (dash) are shown (See FIG. 2).
Example 3
Distribution of Anti-S. pneumoniae Antibody Titers at 10 Weeks
after Three Vaccinations
[0279] FIG. 3 shows Anti-S. pneumoniae IgG titer distributions at
week 10, after three intranasal vaccine doses given one month apart
on weeks 0, 4, and 8 for experimental and control groups. All mice
vaccinated with 10.sup.8 CFU WCPAg plus varying concentrations of
nanoemulsion obtained a serum antibody titer of 5.times.10.sup.3 or
greater, with a maximum of 5.times.10.sup.6 and an average across
all 10.sup.8 CFU/vaccine groups of 4.times.10.sup.5. Titers for
individual animals (circles) and mean serum titer per group (dash)
are shown (See FIG. 3).
Example 4
Nanoemulsion Utilized to Adjuvant Ethanol Killed Whole Cell S.
pneumoniae
[0280] Unless otherwise described herein, materials and methods
indicated in Example 1 were utilized. Ethanol inactivated whole
cell S. pneumoniae antigen (either 10.sup.6 or 10.sup.8 cells) was
mixed with W.sub.805EC nanoemulsion (ranging from 1% to 20%). This
mixture was used to intranasally vaccinate (6 .mu.l/nare) 8 week
old outbred CD-1 mice (Jackson Labs, Bar arbor, ME). The mice were
vaccinated and then boosted at 4 weeks following prime vaccination.
The mice were nasally inoculated with 10.sup.8 live wildtype strain
6B S. pneumoniae at 11 weeks and sacrificed for colony enumeration
at 12 weeks. Serum anti-pneumococcal IgG titers, as measured by
ELISA, were found to be 1 to 1.5 logs greater than negative
controls and approached within 0.5 log of positive (alum) control
(See FIG. 4). The high serum titers in the high antigen dose group
correlated with increased ability to eradicate intranasal
colonization following challenge with wild-type S. pneumoniae (See
FIG. 5).
Example 5
Compositions and Methods Utilizing Nanoemulsion Inactivated/Killed
Whole Cell S. pneumoniae
[0281] Nanoemulsion was utilized to kill and/or inactivate live S.
pneumoniae, which was then subsequently administered to subjects to
generate immune response to nanoemulsion killed S. pneumoniae
compositions. In order to evaluate the microbiocidal activity of
nanoemulsion against S. pneumoniae, 1.times.10.sup.8 live,
acapsular LytA-S. pneumoniae mutant was mixed with varying
concentrations of nanoemulsion (W.sub.805EC, 1%, 5%, 10% or 20%).
The bacteria were incubated with NE for 30 minutes, 1 hour or 3
hours. The nanoemulsion was separated by centrifugation and the
killed/inactivated whole cell acapsular LytA-S. pneumoniae pellets
were washed to remove any remaining NE. Resuspended pellets were
plated on blood agar for colony enumeration. Complete inactivation
of the S. pneumoniae was noted at all time points (See FIG. 6).
[0282] A composition comprising nanoemulsion killed S. pneumoniae
was utilized to generate immune response in subjects. Two different
concentrations of nanoemulsion (5% and 15%) were utilized to
inactivate 10.sup.7 or 10.sup.9 live, acapsular LytA-S. pneumoniae
mutant cells. For comparison, bacteria were also inactivated with
ethanol (EI). Inactivation was verified using plate culture.
[0283] Following the inactivation procedure, mice were immunized
with the combined inactivated S. pneumoniae and NE. Immunizations
were delivered intranasally except for positive (Alum) control. The
mice were primed and then boosted at 4 weeks (See FIG. 7). The mice
were nasally inoculated with 10.sup.8 live wildtype strain 6B S.
pneumoniae at 8 weeks and sacrificed for colony enumeration at 9
weeks. Serum anti-pneumococcal IgG titers were observed that
approached 10.sup.5 in mice vaccinated with 15% NE-10.sup.9
inactivated bacteria. These titers were equivalent or greater than
(alum) control mice (See FIG. 8). The high serum titers in the high
antigen dose group correlated with increased ability to eradicate
intranasal colonization following challenge with wild-type S.
pneumoniae (See FIG. 9).
[0284] Thus, in some embodiments, the present invention provide
that nasal administration of a whole cell Streptococcus pneumoniae
antigen (WCPAg, killed and/or inactivated by mixing with ethanol
and/or nanoemulsion) mixed with nanoemulsion induces IgG response
and eradicates upper respiratory colonization.
Example 6
Identification of S. pneumoniae Immunogens
[0285] Although an understanding of a mechanism is not necessary to
practice the present invention, and the invention is not limited to
any particular mechanism of action, experiments were conducted
during development of embodiments of the invention in order to
further characterize and/or identify immunoreactive proteins
present in inactivated S. pneumoniae, (e.g., inactivated using
nanoemulsion or ethanol).
[0286] Experiments were designed to identify potential protective
antigens. Work involved identification of immunoreactive proteins
via western blotting. NE inactivated S. pneumoniae, ethanol
inactived S. pneumoniae, and wildtype 6B inactivated protein were
electrophoretically separated and probed with either serum from
mice vaccinated with 15% NE-10.sup.9 S. pneumoniae or 0.5 mg/kg
Alum-S. pneumoniae (See FIG. 10). Bands at several molecular
weights corresponding to known conserved or semi-conserved
pneumococcal proteins were identified (e.g., PsaA, PiuA, PavA).
[0287] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described compositions and
methods of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the
present invention.
* * * * *